AIDS Vaccine Clinical Trials
Ake, J. A., A. Schuetz, P. Pegu, L. Wieczorek, M. A. Eller, H. Kibuuka, F. Sawe, L. Maboko, V. Polonis, N. Karasavva, D. Weiner, A. Sekiziyivu, J. Kosgei, M. Missanga, A. Kroidl, P. Mann, S. Ratto-Kim, L. A. Eller, P. Earl, B. Moss, J. Dorsey-Spitz, M. Milazzo, G. L. Ouedraogo, F. Rizvi, J. Yan, A. S. Khan, S. Peel, N. Y. Sardesai, N. L. Michael, V. Ngauy, M. Marovich and M. L. Robb (2017). “Safety and Immunogenicity of PENNVAX(R)-G DNA Prime Administered by Biojector(R) 2000 or CELLECTRA(R) Electroporation Device with Modified Vaccinia Ankara-CMDR Boost.” J Infect Dis.
Background: We report the first-in-human safety and immunogenicity evaluation of PENNVAX(R)-G DNA/ MVA-CMDR prime-boost HIV vaccine, with intramuscular DNA delivery by either Biojector(R) 2000 needle free injection system (Biojector) or by CELLECTRA(R) electroporation device. Methods: Healthy, HIV-uninfected adults were randomized to receive 4 mg PENNVAX(R)-G DNA delivered IM by Biojector or electroporation at baseline and week 4 followed by IM injection of 108 pfu MVA-CMDR at week 12 and 24. The open label Part A was conducted in the US, followed by a double-blind, placebo controlled Part B in East Africa. Solicited and unsolicited adverse events were recorded and immune responses measured. Results: 88/100 enrolled participants completed all study injections, which were generally safe and well tolerated, with more immediate, but transient, pain in the electroporation group. Cellular responses were observed in 57% of vaccine recipients tested and were CD4 predominant. High rates of binding antibody responses to CRF01_AE antigens, including gp70 V1V2 scaffold, were observed. Neutralizing antibodies were detected in a peripheral blood mononuclear cell assay and moderate antibody dependent cell-mediated cytotoxicity activity was demonstrated. Discussion: The PVG/MVA-CMDR HIV-1 vaccine regimen is safe and immunogenic. Substantial differences in safety or immunogenicity between modes of DNA delivery were not observed. Clinical Trials Registration: NCT01260727, https://clinicaltrials.gov/ct2/show/NCT01260727.
Viegas, E. O., N. Tembe, C. Nilsson, B. Meggi, C. Maueia, O. Augusto, R. Stout, G. Scarlatti, G. Ferrari, P. Earl, B. Wahren, S. Andersson, M. Robb, N. Osman, G. Biberfeld, I. Jani and E. Sandstrom (2017). “Intradermal HIV-1 DNA immunization using needle-free Zetajet<sup>TM</sup> injection followed by HIV-modified vaccinia virus Ankara vaccination is safe and immunogenic in Mozambican young adults: a phase I randomized controlled trial.” AIDS Res Hum Retroviruses.
We assessed safety and immunogenicity of HIV-DNA priming using Zetajet<sup>TM</sup>, a needle-free device intradermally followed by intramuscular HIV-MVA boosts, in 24 healthy Mozambicans. Volunteers were randomized to receive three immunizations of 600 microg (n = 10; 2 x 0.1mL) or 1200 microg (n = 10; 2 x 0.2mL) of HIV-DNA (3 mg/mL), followed by two boosts of 10<sup>8</sup>pfu HIV-MVA. Four subjects received placebo saline injections. Vaccines and injections were safe and well tolerated with no difference between the two priming groups. After three HIV-DNA immunizations, IFN-gamma ELISpot responses to Gag were detected in 9/17 (53%) vaccinees, while none responded to Env. After the first HIV-MVA, the overall response rate to Gag and/or Env increased to 14/15 (93%); 14/15 (93%) to Gag and 13/15 (87%) to Env. There were no significant differences between the immunization groups in frequency of response to Gag and Env or magnitude of Gag responses. Env responses were significantly higher in the higher-dose group (median 420 vs 157.5 SFC/million PBMC, p=0.014). HIV-specific antibodies to subtype C gp140 and subtype B gp160 were elicited in all vaccinees after the second HIV-MVA, without differences in titers between the groups. Neutralizing antibody responses were not detected. Two (13%) of 16 vaccinees, one in each of the priming groups, exhibited antibodies mediating antibody-dependent cellular cytotoxicity to CRF01_AE. In conclusion, HIV-DNA vaccine delivered intradermally in volumes of 0.1-0.2 mL using Zetajet<sup>TM</sup> was safe and well tolerated. Priming with the 1200 microg dose of HIV-DNA generated higher magnitudes of ELISpot responses to Env.
AIDS Vaccine Design Immunogenicity Efficacy
Wang, N., Z. Yuan, W. Niu, Q. Li and J. Guo (2017). “Synthetic biology approach for the development of conditionally replicating HIV-1 vaccine.” J Chem Technol Biotechnol 92(3): 455-462.
While the combined antiretroviral therapy has resulted in a significant decrease in HIV-1 related morbidity and mortality, the HIV-1 pandemic has not been substantially averted. To curtail the 2.4 million new infections each year, a prophylactic HIV-1 vaccine is urgently needed. This review first summarizes four major completed clinical efficacy trials of prophylactic HIV-1 vaccine and their outcomes. Next, it discusses several other approaches that have not yet advanced to clinical efficacy trials, but provided valuable insights into vaccine design. Among them, live-attenuated vaccines (LAVs) provided excellent protection in a non-human primate model. However, safety concerns have precluded the current version of LAVs from clinical application. As the major component of this review, two synthetic biology approaches for improving the safety of HIV-1 LAVs through controlling HIV-1 replication are discussed. Particular focus is on a novel approach that uses unnatural amino acid-mediated suppression of amber nonsense codon to generate conditionally replicating HIV-1 variants. The objective is to attract more attention towards this promising research field and to provoke creative designs and innovative utilization of the two control strategies.
AIDS Vaccine Envelope Design
Gift, S. K., D. P. Leaman, L. Zhang, A. S. Kim and M. B. Zwick (2017). “Functional Stability of HIV-1 Envelope Trimer Affects Accessibility to Broadly Neutralizing Antibodies at its Apex.” J Virol.
The trimeric envelope glycoprotein spike (Env) of HIV-1 is the target of vaccine development to elicit broadly neutralizing antibodies (bnAbs). Env trimer instability and heterogeneity in principle make subunit interfaces inconsistent targets for the immune response. Here, we investigate how functional stability of Env relates to neutralization sensitivity to V2 bnAbs and V3 crown antibodies that engage subunit interfaces upon binding to unliganded Env. Env heterogeneity was inferred when antibodies neutralized a mutant Env with a plateau of less than 100% percentage neutralization. A statistically significant correlation was found between the stability of mutant Envs and the MPN of V2 bnAb, PG9, as well as an inverse correlation between stability of Env and neutralization by V3 crown antibody, 447-52D. A number of Env-stabilizing mutations and V2 bnAb-enhancing mutations were identified in Env, but did not always overlap indicating distinct requirements of functional stabilization versus antibody recognition. Blocking complex glycosylation of Env affected V2 bnAb recognition, as previously described, but also notably increased functional stability of Env. This study shows how instability and heterogeneity affect antibody sensitivity of HIV-1 Env, which is relevant to vaccine design involving its dynamic apex. IMPORTANCE: The Env trimer is the only viral protein on the surface of HIV-1 and is the target of neutralizing antibodies that reduce viral infectivity. Quaternary epitopes at the apex of the spike are recognized by some of the most potent and broadly neutralizing antibodies to date. Being that their glycan-protein hybrid epitopes are at subunit interfaces, the resulting heterogeneity can lead to partial neutralization. Here, we screened for mutations in Env that allowed for complete neutralization by the bnAbs. We found that when mutations outside of V2 increased V2 bnAb recognition they often also increased Env stability-of-function and decreased binding by narrowly neutralizing antibodies to the V3 crown. Three mutations together increased neutralization by V2 bnAb and eliminated binding by V3 crown antibodies. These results may aid the design of immunogens that elicit antibodies to the trimer apex.
Gristick, H. B., H. Wang and P. J. Bjorkman (2017). “X-ray and EM structures of a natively glycosylated HIV-1 envelope trimer.” Acta Crystallogr D Struct Biol 73(Pt 10): 822-828.
The structural and biochemical characterization of broadly neutralizing anti-HIV-1 antibodies (bNAbs) has been essential in guiding the design of potential vaccines to prevent infection by HIV-1. While these studies have revealed critical mechanisms by which bNAbs recognize and/or accommodate N-glycans on the trimeric envelope glycoprotein (Env), they have been limited to the visualization of high-mannose glycan forms only, since heterogeneity introduced from the presence of complex glycans makes it difficult to obtain high-resolution structures. 3.5 and 3.9 A resolution crystal structures of the HIV-1 Env trimer with fully processed and native glycosylation were solved, revealing a glycan shield of high-mannose and complex-type N-glycans that were used to define the complete epitopes of two bNAbs. Here, the refinement of the N-glycans in the crystal structures is discussed and comparisons are made with glycan densities in glycosylated Env structures derived by single-particle cryo-electron microscopy.
Karlsson, I., M. Borggren, S. S. Jensen, L. Heyndrickx, G. Stewart-Jones, G. Scarlatti and A. Fomsgaard (2017). “Immunization with clinical HIV-1 Env proteins induces broad antibody-dependent cellular cytotoxicity (ADCC)-mediating antibodies in a rabbit vaccination model.” AIDS Res Hum Retroviruses.
The induction of both neutralizing antibodies and non-neutralizing antibodies with effector functions, e.g., antibody-dependent cellular cytotoxicity (ADCC), is desired in the search for effective vaccines against HIV-1. In the pursuit of novel immunogens capable of inducing an efficient antibody response, rabbits were immunized with selected antigens using different prime-boost strategies. We immunized 35 different groups of rabbits with Env antigens from clinical HIV-1 subtypes A and B, including immunization with DNA alone, protein alone and DNA prime with protein boost. The rabbit sera were screened for ADCC activity using a GranToxiLux-based assay with human PBMCs as effector cells and CEM.NKRCCR5 cells coated with HIV-1 envelope as target cells. The groups with the highest ADCC activity were further characterized for cross-reactivity between HIV-1 subtypes. The immunogen inducing the most potent and broadest ADCC response was a trimeric gp140. The ADCC activity was highest against the HIV-1 subtype corresponding to the immunogen. The ADCC activity did not necessarily reflect neutralizing activity in the pseudovirus-TZMbl assay, but there was an overall correlation between the two antiviral activities. We present a rabbit vaccination model and an assay suitable for screening HIV-1 vaccine candidates for the induction of ADCC-mediating antibodies in addition to neutralizing antibodies. The antigens and/or immunization strategies capable of inducing antibodies with ADCC activity did not necessarily induce neutralizing activity, and vice versa. Nevertheless, we identified vaccine candidates that were able to concurrently induce both types of responses and that had ADCC activity that was cross-reactive between different subtypes. When searching for an effective vaccine candidate, it is important to evaluate the antibody response using a model and an assay measuring the desired function.
Molinos-Albert, L. M., B. Clotet, J. Blanco and J. Carrillo (2017). “Immunologic Insights on the Membrane Proximal External Region: A Major Human Immunodeficiency Virus Type-1 Vaccine Target.” Front Immunol 8: 1154.
Broadly neutralizing antibodies (bNAbs) targeting conserved regions within the human immunodeficiency virus type-1 (HIV-1) envelope glycoprotein (Env) can be generated by the human immune system and their elicitation by vaccination will be a key point to protect against the wide range of viral diversity. The membrane proximal external region (MPER) is a highly conserved region within the Env gp41 subunit, plays a major role in membrane fusion and is targeted by naturally induced bNAbs. Therefore, the MPER is considered as an attractive vaccine target. However, despite many attempts to design MPER-based immunogens, further study is still needed to understand its structural complexity, its amphiphilic feature, and its limited accessibility by steric hindrance. These particular features compromise the development of MPER-specific neutralizing responses during natural infection and limit the number of bNAbs isolated against this region, as compared with other HIV-1 vulnerability sites, and represent additional hurdles for immunogen development. Nevertheless, the analysis of MPER humoral responses elicited during natural infection as well as the MPER bNAbs isolated to date highlight that the human immune system is capable of generating MPER protective antibodies. Here, we discuss the recent advances describing the immunologic and biochemical features that make the MPER a unique HIV-1 vulnerability site, the different strategies to generate MPER-neutralizing antibodies in immunization protocols and point the importance of extending our knowledge toward new MPER epitopes by the isolation of novel monoclonal antibodies. This will be crucial for the redesign of immunogens able to skip non-neutralizing MPER determinants.
Kennedy, S. B., F. Bolay, M. Kieh, G. Grandits, M. Badio, R. Ballou, R. Eckes, M. Feinberg, D. Follmann, B. Grund, S. Gupta, L. Hensley, E. Higgs, K. Janosko, M. Johnson, F. Kateh, J. Logue, J. Marchand, T. Monath, M. Nason, T. Nyenswah, F. Roman, E. Stavale, J. Wolfson, J. D. Neaton, H. C. Lane and P. I. S. Group (2017). “Phase 2 Placebo-Controlled Trial of Two Vaccines to Prevent Ebola in Liberia.” N Engl J Med 377(15): 1438-1447.
Background: The safety and efficacy of vaccines to prevent Ebola virus disease (EVD) were unknown when the incidence of EVD was peaking in Liberia. Methods We initiated a randomized, placebo-controlled, phase 3 trial of the chimpanzee adenovirus 3 vaccine (ChAd3-EBO-Z) and the recombinant vesicular stomatitis virus vaccine (rVSVG-ZEBOV-GP) in Liberia. A phase 2 subtrial was embedded to evaluate safety and immunogenicity. Because the incidence of EVD declined in Liberia, the phase 2 component was expanded and the phase 3 component was eliminated. Results A total of 1500 adults underwent randomization and were followed for 12 months. The median age of the participants was 30 years; 36.6% of the participants were women. During the week after the administration of vaccine or placebo, adverse events occurred significantly more often with the active vaccines than with placebo; these events included injection-site reactions (in 28.5% of the patients in the ChAd3-EBO-Z group and 30.9% of those in the rVSVG-ZEBOV-GP group, as compared with 6.8% of those in the placebo group), headache (in 25.1% and 31.9%, vs. 16.9%), muscle pain (in 22.3% and 26.9%, vs. 13.3%), feverishness (in 23.9% and 30.5%, vs. 9.0%), and fatigue (in 14.0% and 15.4%, vs. 8.8%) (P<0.001 for all comparisons); these differences were not seen at 1 month. Serious adverse events within 12 months after injection were seen in 40 participants (8.0%) in the ChAd3-EBO-Z group, in 47 (9.4%) in the rVSVG-ZEBOV-GP group, and in 59 (11.8%) in the placebo group. By 1 month, an antibody response developed in 70.8% of the participants in the ChAd3-EBO-Z group and in 83.7% of those in the rVSVG-ZEBOV-GP group, as compared with 2.8% of those in the placebo group (P<0.001 for both comparisons). At 12 months, antibody responses in participants in the ChAd3-EBO-Z group (63.5%) and in those in the rVSVG-ZEBOV-GP group (79.5%) remained significantly greater than in those in the placebo group (6.8%, P<0.001 for both comparisons). Conclusions A randomized, placebo-controlled phase 2 trial of two vaccines that was rapidly initiated and completed in Liberia showed the capability of conducting rigorous research during an outbreak. By 1 month after vaccination, the vaccines had elicited immune responses that were largely maintained through 12 months. (Funded by the National Institutes of Allergy and Infectious Diseases and the Liberian Ministry of Health; PREVAIL I ClinicalTrials.gov number, NCT02344407 .).
Agnandji, S. T., J. F. Fernandes, E. B. Bache, R. M. Obiang Mba, J. S. Brosnahan, L. Kabwende, P. Pitzinger, P. Staarink, M. Massinga-Loembe, V. Krahling, N. Biedenkopf, S. K. Fehling, T. Strecker, D. J. Clark, H. M. Staines, J. W. Hooper, P. Silvera, V. Moorthy, M. P. Kieny, A. A. Adegnika, M. P. Grobusch, S. Becker, M. Ramharter, B. Mordmuller, B. Lell, S. Krishna and P. G. Kremsner (2017). “Safety and immunogenicity of rVSVDeltaG-ZEBOV-GP Ebola vaccine in adults and children in Lambarene, Gabon: A phase I randomised trial.”PLoS Med 14(10): e1002402.
BACKGROUND: The rVSVDeltaG-ZEBOV-GP vaccine prevented Ebola virus disease when used at 2 x 107 plaque-forming units (PFU) in a trial in Guinea. This study provides further safety and immunogenicity data. METHODS AND FINDINGS: A randomised, open-label phase I trial in Lambarene, Gabon, studied 5 single intramuscular vaccine doses of 3 x 103, 3 x 104, 3 x 105, 3 x 106, or 2 x 107 PFU in 115 adults and a dose of 2 x 107 PFU in 20 adolescents and 20 children. The primary objective was safety and tolerability 28 days post-injection. Immunogenicity, viraemia, and shedding post-vaccination were evaluated as secondary objectives. In adults, mild-to-moderate adverse events were frequent, but there were no serious or severe adverse events related to vaccination. Before vaccination, Zaire Ebola virus (ZEBOV)-glycoprotein (GP)-specific and ZEBOV antibodies were detected in 11% and 27% of adults, respectively. In adults, 74%-100% of individuals who received a dose 3 x 104, 3 x 105, 3 x 106, or 2 x 107 PFU had a >/=4.0-fold increase in geometric mean titres (GMTs) of ZEBOV-GP-specific antibodies at day 28, reaching GMTs of 489 (95% CI: 264-908), 556 (95% CI: 280-1,101), 1,245 (95% CI: 899-1,724), and 1,503 (95% CI: 931-2,426), respectively. Twenty-two percent of adults had a >/=4-fold increase of ZEBOV antibodies, with GMTs at day 28 of 1,015 (647-1,591), 1,887 (1,154-3,085), 1,445 (1,013-2,062), and 3,958 (2,249-6,967) for the same doses, respectively. These antibodies persisted up to day 180 for doses >/=3 x 105 PFU. Adults with antibodies before vaccination had higher GMTs throughout. Neutralising antibodies were detected in more than 50% of participants at doses >/=3 x 105 PFU. As in adults, no serious or severe adverse events related to vaccine occurred in adolescents or children. At day 2, vaccine RNA titres were higher for adolescents and children than adults. At day 7, 78% of adolescents and 35% of children had recombinant vesicular stomatitis virus RNA detectable in saliva. The vaccine induced high GMTs of ZEBOV-GP-specific antibodies at day 28 in adolescents, 1,428 (95% CI: 1,025-1,989), and children, 1,620 (95% CI: 806-3,259), and in both groups antibody titres increased up to day 180. The absence of a control group, lack of stratification for baseline antibody status, and imbalances in male/female ratio are the main limitations of this study. CONCLUSIONS: Our data confirm the acceptable safety and immunogenicity profile of the 2 x 107 PFU dose in adults and support consideration of lower doses for paediatric populations and those who request boosting. TRIAL REGISTRATION: Pan African Clinical Trials Registry PACTR201411000919191.
Emerging Infectious Diseases
Carrasco-Hernandez, R., R. Jacome, Y. Lopez Vidal and S. Ponce de Leon (2017). “Are RNA Viruses Candidate Agents for the Next Global Pandemic? A Review.” Ilar j: 1-16.
Pathogenic RNA viruses are potentially the most important group involved in zoonotic disease transmission, and they represent a challenge for global disease control. Their biological diversity and rapid adaptive rates have proved to be difficult to overcome and to anticipate by modern medical technology. Also, the anthropogenic change of natural ecosystems and the continuous population growth are driving increased rates of interspecies contacts and the interchange of pathogens that can develop into global pandemics. The combination of molecular, epidemiological, and ecological knowledge of RNA viruses is therefore essential towards the proper control of these emergent pathogens. This review outlines, throughout different levels of complexity, the problems posed by RNA viral diseases, covering some of the molecular mechanisms allowing them to adapt to new host species-and to novel pharmaceutical developments-up to the known ecological processes involved in zoonotic transmission.
Grifoni, A., J. Pham, J. Sidney, P. H. O’Rourke, S. Paul, B. Peters, S. R. Martini, A. D. de Silva, M. J. Ricciardi, D. M. Magnani, C. G. T. Silveira, A. Maestri, P. R. Costa, L. M. de-Oliveira-Pinto, E. L. de Azeredo, P. V. Damasco, E. Phillips, S. Mallal, A. M. de Silva, M. Collins, A. Durbin, S. A. Diehl, C. Cerpas, A. Balmaseda, G. Kuan, J. Coloma, E. Harris, J. E. Crowe, Jr., M. Stone, P. J. Norris, M. Busch, H. Vivanco-Cid, J. Cox, B. S. Graham, J. E. Ledgerwood, L. Turtle, T. Solomon, E. G. Kallas, D. I. Watkins, D. Weiskopf and A. Sette (2017). “Prior Dengue virus exposure shapes T cell immunity to Zika virus in humans.” J Virol.
While progress has been made in characterizing humoral immunity to Zika virus (ZIKV) in humans, little is known regarding the corresponding T cell responses to ZIKV. Here we investigate the kinetics and viral epitopes targeted by T cells responding to ZIKV and address the critical question of whether pre-existing dengue virus (DENV) T cell immunity modulates these responses. We find that memory T cell responses elicited by prior infection with DENV or vaccination with Tetravalent Dengue Attenuated Vaccines (TDLAV) recognize ZIKV-derived peptides. This cross-reactivity is explained by the sequence similarity of the two viruses, as the ZIKV peptides recognized by DENV-elicited memory T cells are identical or highly conserved in DENV and ZIKV. DENV exposure prior to ZIKV infection also influences the timing and magnitude of the T cell response. ZIKV-reactive T cells in the acute phase of infection are detected earlier and in greater magnitude in DENV-immune patients. Conversely, the frequency of ZIKV-reactive T cells continues to rise in the convalescent phase in DENV-naive donors, but declines in DENV pre-exposed donors, compatible with more efficient control of ZIKV replication and/or clearance of ZIKV antigen. The quality of responses is also influenced by previous DENV exposure, and ZIKV-specific CD8 T cells form DENV pre-exposed donors selectively up-regulated granzyme B and PD1, as compared to DENV-naive donors. Finally, we discovered that ZIKV structural proteins (E, prM and C) are major targets of both the CD4 and CD8 T cell responses, whereas DENV T cell epitopes are found primarily in nonstructural proteins. IMPORTANCE: The issue of potential ZIKV and DENV cross-reactivity and how pre-existing DENV T cell immunity modulates ZIKA T cell responses is of great relevance as the two viruses often co-circulate and ZIKA virus has been spreading in geographical regions where DENV is endemic or hyper-endemic. Our data show that memory T cell responses elicited by prior infection with DENV recognize ZIKV-derived peptides and that DENV exposure prior to ZIKV infection influences the timing, magnitude and quality of the T cell response. Additionally we show that ZIKV-specific responses target different proteins than DENV-specific responses, pointing towards important implications for vaccine design against this global threat.
Emerging Infectious Diseases Vaccine Design
Magnani, D. M., T. F. Rogers, N. Beutler, M. J. Ricciardi, V. K. Bailey, L. Gonzalez-Nieto, B. Briney, D. Sok, K. Le, A. Strubel, M. J. Gutman, N. Pedreno-Lopez, N. D. Grubaugh, C. G. T. Silveira, H. S. Maxwell, A. Domingues, M. A. Martins, D. E. Lee, E. E. Okwuazi, S. Jean, E. A. Strobert, A. Chahroudi, G. Silvestri, T. H. Vanderford, E. G. Kallas, R. C. Desrosiers, M. C. Bonaldo, S. S. Whitehead, D. R. Burton and D. I. Watkins (2017). “Neutralizing human monoclonal antibodies prevent Zika virus infection in macaques.” Sci Transl Med 9(410).
Therapies to prevent maternal Zika virus (ZIKV) infection and its subsequent fetal developmental complications are urgently required. We isolated three potent ZIKV-neutralizing monoclonal antibodies (nmAbs) from the plasmablasts of a ZIKV-infected patient-SMZAb1, SMZAb2, and SMZAb5-directed against two different domains of the virus. We engineered these nmAbs with Fc LALA mutations that abrogate Fcgamma receptor binding, thus eliminating potential therapy-mediated antibody-dependent enhancement. We administered a cocktail of these three nmAbs to nonhuman primates 1 day before challenge with ZIKV and demonstrated that the nmAbs completely prevented viremia in serum after challenge. Given that numerous antibodies have exceptional safety profiles in humans, the cocktail described here could be rapidly developed to protect uninfected pregnant women and their fetuses.
Mire, C. E., R. W. Cross, J. B. Geisbert, V. Borisevich, K. N. Agans, D. J. Deer, M. L. Heinrich, M. M. Rowland, A. Goba, M. Momoh, M. L. Boisen, D. S. Grant, M. Fullah, S. H. Khan, K. A. Fenton, J. E. Robinson, L. M. Branco, R. F. Garry and T. W. Geisbert (2017). “Human-monoclonal-antibody therapy protects nonhuman primates against advanced Lassa fever.” Nat Med 23(10): 1146-1149.
There are no approved treatments for Lassa fever, which is endemic to the same regions of West Africa that were recently devastated by Ebola. Here we show that a combination of human monoclonal antibodies that cross-react with the glycoproteins of all four clades of Lassa virus is able to rescue 100% of cynomolgus macaques when treatment is initiated at advanced stages of disease, including up to 8 d after challenge.
Tebas, P., C. C. Roberts, K. Muthumani, E. L. Reuschel, S. B. Kudchodkar, F. I. Zaidi, S. White, A. S. Khan, T. Racine, H. Choi, J. Boyer, Y. K. Park, S. Trottier, C. Remigio, D. Krieger, S. E. Spruill, M. Bagarazzi, G. P. Kobinger, D. B. Weiner and J. N. Maslow (2017). “Safety and Immunogenicity of an Anti-Zika Virus DNA Vaccine – Preliminary Report.” N Engl J Med.
Background Although Zika virus (ZIKV) infection is typically self-limiting, other associated complications such as congenital birth defects and the Guillain-Barre syndrome are well described. There are no approved vaccines against ZIKV infection. Methods In this phase 1, open-label clinical trial, we evaluated the safety and immunogenicity of a synthetic, consensus DNA vaccine (GLS-5700) encoding the ZIKV premembrane and envelope proteins in two groups of 20 participants each. The participants received either 1 mg or 2 mg of vaccine intradermally, with each injection followed by electroporation (the use of a pulsed electric field to introduce the DNA sequence into cells) at baseline, 4 weeks, and 12 weeks. Results The median age of the participants was 38 years, and 60% were women; 78% were white, and 22% black; in addition, 30% were Hispanic. At the interim analysis at 14 weeks (i.e., after the third dose of vaccine), no serious adverse events were reported. Local reactions at the vaccination site (e.g., injection-site pain, redness, swelling, and itching) occurred in approximately 50% of the participants. After the third dose of vaccine, binding antibodies (as measured on enzyme-linked immunosorbent assay) were detected in all the participants, with geometric mean titers of 1642 and 2871 in recipients of 1 mg and 2 mg of vaccine, respectively. Neutralizing antibodies developed in 62% of the samples on Vero-cell assay. On neuronal-cell assay, there was 90% inhibition of ZIKV infection in 70% of the serum samples and 50% inhibition in 95% of the samples. The intraperitoneal injection of postvaccination serum protected 103 of 112 IFNAR knockout mice (bred with deletion of genes encoding interferon-alpha and interferon-beta receptors) (92%) that were challenged with a lethal dose of ZIKV-PR209 strain; none of the mice receiving baseline serum survived the challenge. Survival was independent of the neutralization titer. Conclusions In this phase 1, open-label clinical trial, a DNA vaccine elicited anti-ZIKV immune responses. Further studies are needed to better evaluate the safety and efficacy of the vaccine. (Funded by GeneOne Life Science and others; ZIKA-001 ClinicalTrials.gov number, NCT02809443 .).
Urakami, A., M. M. Ngwe Tun, M. L. Moi, A. Sakurai, M. Ishikawa, S. Kuno, R. Ueno, K. Morita and W. Akahata (2017). “Envelope-modified tetravalent dengue virus-like particle vaccine: implication for flavivirus vaccine design.” J Virol.
Dengue viruses (DENV) infect 50-100 million people each year. The spread of DENV-associated infections is one of the most serious public health problems worldwide, as there is no widely available vaccine or specific therapeutic for DENV infections. To address this, we developed a novel tetravalent dengue vaccine utilizing virus-like particle (VLP) technology. We created recombinant DENV1-4 VLPs by co-expressing precursor membrane (prM) and envelope (E) proteins, with a F108A mutation in the fusion loop structure of E to increase the production of VLPs in mammalian cells. Immunization with DENV1-4 VLPs as individual, monovalent vaccines elicited strong neutralization activity against each DENV serotype in mice. When immunized as a tetravalent vaccine, DENV1-4 VLPs elicited high levels of neutralization activity against all four serotypes simultaneously. The neutralization antibody response induced by the VLPs was significantly higher than DNA or recombinant E proteins immunization. Moreover, antibody-dependent enhancement (ADE) was not observed against any serotype at 1:10 serum dilution. We also demonstrated that Zika virus (ZIKV) VLP production level was enhanced by introducing the same F108A mutation in ZIKV envelope protein. Taken together, these results suggest that our strategy for DENV VLP production is applicable to other flavivirus VLP vaccine development, due to the similarity in their viral structures and describes the promising development of an effective tetravalent vaccine against the prevalent flavivirus. Importance: The dengue virus poses one of the most serious public health problems worldwide, and the incidence of diseases caused by the virus has increased dramatically. Despite decades of effort, there is no effective treatment against dengue. A safe and potent vaccine against dengue is still needed. We have developed a novel tetravalent dengue vaccine using virus-like particle (VLP) technology, which is non-infectious as it lacks viral genome. Previous attempts by other groups with dengue virus VLPs resulted in generally poor yields. We found that a critical amino acid mutation in the envelope protein enhances the production of VLP. Our tetravalent vaccine elicited potent neutralizing antibody responses against all four serotypes. Our finding can also be applied to vaccine development against other flaviviruses, such as Zika virus or West Nile virus.
HIV – Africa
Dong, K. L., A. Moodley, D. S. Kwon, M. S. Ghebremichael, M. Dong, N. Ismail, Z. M. Ndhlovu, J. M. Mabuka, D. M. Muema, K. Pretorius, N. Lin, B. D. Walker and T. Ndung’u (2017). “Detection and treatment of Fiebig stage I HIV-1 infection in young at-risk women in South Africa: a prospective cohort study.” Lancet HIV.
BACKGROUND: HIV incidence among young women in sub-Saharan Africa remains high and their inclusion in vaccine and cure efforts is crucial. We aimed to establish a cohort of young women detected during Fiebig stage I acute HIV infection in whom treatment was initiated immediately after diagnosis to advance research in this high-risk group. METHODS: 945 women aged 18-23 years in KwaZulu-Natal, South Africa, who were HIV uninfected and sexually active consented to HIV-1 RNA testing twice a week and biological sampling and risk assessment every 3 months during participation in a 48-96 week life-skills and job-readiness programme. We analysed the effect of immediate combination antiretroviral therapy (ART) on viraemia and immune responses, sexual risk behaviour, and the effect of the socioeconomic intervention. FINDINGS: 42 women were diagnosed with acute HIV infection between Dec 1, 2012, and June 30, 2016, (incidence 8.2 per 100 person-years, 95% CI 5.9-11.1), of whom 36 (86%) were diagnosed in Fiebig stage I infection with a median initial viral load of 2.97 log10 copies per mL (IQR 2.42-3.85). 23 of these 36 women started ART at a median of 1 day (1-1) after detection, which limited the median peak viral load to 4.22 log10 copies per mL (3.27-4.83) and the CD4 nadir to 685 cells per muL (561-802). ART also suppressed viral load (to <20 copies per mL) within a median of 16 days (12-26) and, in 20 (87%) of 23 women, prevented seroconversion, as shown with western blotting. 385 women completed the 48 week socioeconomic intervention, of whom 231 were followed up for 1 year. 202 (87%) of these 231 women were placed in jobs, returned to school, or started a business. INTERPRETATION: Frequent HIV screening combined with a socioeconomic intervention facilitated sampling and risk assessment before and after infection. In addition to detection of acute infection and immediate treatment, we established a cohort optimised for prevention and cure research. FUNDING: Bill & Melinda Gates Foundation, National Institute of Allergy and Infectious Diseases, International AIDS Vaccine Initiative, Wellcome Trust, Howard Hughes Medical Institute.
Gumede-Moyo, S., S. Filteau, T. Munthali, J. Todd and P. Musonda (2017). “Implementation effectiveness of revised (post-2010) World Health Organization guidelines on prevention of mother-to-child transmission of HIV using routinely collected data in sub-Saharan Africa: A systematic literature review.” Medicine (Baltimore) 96(40): e8055.
BACKGROUND: To synthesize and evaluate the impact of implementing post-2010 World Health Organization (WHO) prevention of mother-to-child transmission (PMTCT) guidelines on attainment of PMTCT targets. METHODS: Retrospective and prospective cohort study designs that utilized routinely collected data with a focus on provision and utilization of the cascade of PMTCT services were included. The outcomes included the proportion of pregnant women who were tested during their antenatal clinic (ANC) visits; mother-to-child transmission (MTCT) rate; adherence; retention rate; and loss to follow-up (LTFU). RESULTS: Of the 1210 references screened, 45 met the inclusion criteria. The studies originated from 14 countries in sub-Saharan Africa. The highest number of studies originated from Malawi (10) followed by Nigeria and South Africa with 7 studies each. More than half of the studies were on option A while the majority of option B+ studies were conducted in Malawi. These studies indicated a high uptake of human immunodeficiency virus (HIV) testing ranging from 75% in Nigeria to over 96% in Zimbabwe and South Africa. High proportions of CD4 count testing were reported in studies only from South Africa despite that in most of the countries CD4 testing was a prerequisite to access treatment. MTCT rate ranged from 1.1% to 15.1% and it was higher in studies where data were collected in the early days of the WHO 2010 PMTCT guidelines. During the postpartum period, adherence and retention rate decreased, and LTFU increased for both HIV-positive mothers and exposed infants. CONCLUSION: Irrespective of which option was followed, uptake of antenatal HIV testing was high but there was a large drop off along later points in the PMTCT cascade. More research is needed on how to improve later components of the PMTCT cascade, especially of option B+ which is now the norm throughout sub-Saharan Africa.
HIV – Cures & Treatments
Estes, J. D., C. Kityo, F. Ssali, L. Swainson, K. N. Makamdop, G. Q. Del Prete, S. G. Deeks, P. A. Luciw, J. G. Chipman, G. J. Beilman, T. Hoskuldsson, A. Khoruts, J. Anderson, C. Deleage, J. Jasurda, T. E. Schmidt, M. Hafertepe, S. P. Callisto, H. Pearson, T. Reimann, J. Schuster, J. Schoephoerster, P. Southern, K. Perkey, L. Shang, S. W. Wietgrefe, C. V. Fletcher, J. D. Lifson, D. C. Douek, J. M. McCune, A. T. Haase and T. W. Schacker (2017). “Defining total-body AIDS-virus burden with implications for curative strategies.” Nat Med.
In the quest for a functional cure or the eradication of HIV infection, it is necessary to know the sizes of the reservoirs from which infection rebounds after treatment interruption. Thus, we quantified SIV and HIV tissue burdens in tissues of infected nonhuman primates and lymphoid tissue (LT) biopsies from infected humans. Before antiretroviral therapy (ART), LTs contained >98% of the SIV RNA+ and DNA+ cells. With ART, the numbers of virus (v) RNA+ cells substantially decreased but remained detectable, and their persistence was associated with relatively lower drug concentrations in LT than in peripheral blood. Prolonged ART also decreased the levels of SIV- and HIV-DNA+ cells, but the estimated size of the residual tissue burden of 108 vDNA+ cells potentially containing replication-competent proviruses, along with evidence of continuing virus production in LT despite ART, indicated two important sources for rebound following treatment interruption. The large sizes of these tissue reservoirs underscore challenges in developing ‘HIV cure’ strategies targeting multiple sources of virus production.
Huang, Y., G. Pantaleo, G. Tapia, B. Sanchez, L. Zhang, M. Trondsen, A. O. Hovden, R. Pollard, J. Rockstroh, M. Okvist and M. A. Sommerfelt (2017). “Cell-Mediated Immune Predictors of Vaccine Effect on Viral Load and CD4 Count in a Phase 2 Therapeutic HIV-1 Vaccine Clinical Trial.” EBioMedicine.
BACKGROUND: In a placebo-controlled trial of the peptide-based therapeutic HIV-1 p24Gag vaccine candidate Vacc-4x, participants on combination antiretroviral therapy (cART) received six immunizations over 18weeks, followed by analytical treatment interruption (ATI) between weeks 28 and 52. Cell-mediated immune responses were investigated as predictors of Vacc-4x effect (VE) on viral load (VL) and CD4 count during ATI. METHODS: All analyses of week 28 responses and fold-changes relative to baseline considered per-protocol participants (Vacc-4x:placebo=72:32) resuming cART after week 40. Linear regression models with interaction tests were used. VE was estimated as the Vacc-4x-placebo difference in log10-transformed VL (VEVL) or CD4 count (VECD4). FINDINGS: A lower fold-change of CD4+ T-cell proliferation was associated with VECD4 at week 48 (p=0.036, multiplicity adjusted q=0.036) and week 52 (p=0.040, q=0.080). A higher fold-change of IFN-gamma in proliferation supernatants was associated with VEVL at week 44 (p=0.047, q=0.07). A higher fold-change of TNF-alpha was associated with VEVL at week 44 (p=0.045, q=0.070), week 48 (p=0.028, q=0.070), and week 52 (p=0.037, q=0.074). A higher fold-change of IL-6 was associated with VEVL at week 48 (p=0.017, q=0.036). TNF-alpha levels (>median) were associated with VECD4 at week 48 (p=0.009, q=0.009). INTERPRETATION: These exploratory analyses highlight the potential value of investigating biomarkers in T-cell proliferation supernatants for VE in clinical studies.
HIV- Elite Controllers and Exposed Seronegative
Grabar, S., H. Selinger-Leneman, S. Abgrall, G. Pialoux, L. Weiss and D. Costagliola (2017). “Loss of long-term non-progressor and HIV controller status over time in the French Hospital Database on HIV – ANRS CO4.” PLoS One 12(10): e0184441.
OBJECTIVES: We studied the frequency and risk factors for loss of long-term non-progressor (LTNP) and HIV controller (HIC) status among patients identified as such in 2005 in the French Hospital Database on HIV (FHDH-ANRS CO4). METHODS: We selected patients who were treatment-naive and asymptomatic in 2005 (baseline). Those with >/=8 years of known HIV infection and a CD4 cell nadir >/=500/mm3 were classified as LTNP and those with >/=10 years of known HIV infection and 90% of plasma viral load (VL) values </=500 copies/ml in the absence of cART as HIC. cART initiation without loss of status and death from non AIDS-defining causes were considered as competing events. RESULTS: After 5 years of follow-up, 33% (95%CI; 27-42) of 171 LTNP patients and 17% (95%CI; 10-30) of 72 HIC patients had lost their status. In multivariable analyses, loss of LTNP status was associated with lower baseline CD4 cell counts and CD4/CD8 ratios. Only VL was significantly associated with loss of HIC status after adjustment for the baseline CD4 cell count, the CD4/CD8 ratio, and concomitant LTNP status. The hazard ratio for loss of HIC status was 5.5 (95%CI, 1.5-20.1) for baseline VL 50-500 vs </=50 cp/mL, after adjustment for the baseline CD4 cell count. CONCLUSIONS: One-third of LTNP and one-fifth of HIC patients lost their status after 5 years of follow-up, raising questions as to the possible benefits and timing of ART initiation in these populations.
Mukhopadhyay, M., M. Galperin, M. Patgaonkar, S. Vasan, D. D. Ho, A. Nouel, M. Claireaux, D. Benati, O. Lambotte, Y. Huang and L. A. Chakrabarti (2017). “DNA Vaccination by Electroporation Amplifies Broadly Cross-Restricted Public TCR Clonotypes Shared with HIV Controllers.” J Immunol.
Rare patients who spontaneously control HIV replication provide a useful model to inform HIV vaccine development. HIV controllers develop particularly efficient antiviral CD4+ T cell responses mediated by shared high-affinity TCRs. To determine whether the candidate DNA vaccine ADVAX could induce similar responses, we analyzed Gag-specific primary CD4+ T cells from healthy volunteers who received ADVAX DNA by electroporation. Vaccinated volunteers had an immunodominant response to the Gag293 epitope with a functional avidity intermediate between that of controllers and treated patients. The TCR repertoire of Gag293-specific CD4+ T cells proved highly biased, with a predominant usage of the TCRbeta variable gene 2 (TRBV2) in vaccinees as well as controllers. TCRalpha variable gene (TRAV) gene usage was more diverse, with the dominance of TRAV29 over TRAV24 genes in vaccinees, whereas TRAV24 predominated in controllers. Sequence analysis revealed an unexpected degree of overlap between the specific repertoires of vaccinees and controllers, with the sharing of TRAV24 and TRBV2 public motifs (>30%) and of public clonotypes characteristic of high-affinity TCRs. MHC class II tetramer binding revealed a broad HLA-DR cross-restriction, explaining how Gag293-specific public clonotypes could be selected in individuals with diverse genetic backgrounds. TRAV29 clonotypes also proved cross-restricted, but conferred responses of lower functional avidity upon TCR transfer. In conclusion, DNA vaccination by electroporation primed for TCR clonotypes that were associated with HIV control, highlighting the potential of this vaccine delivery method. To our knowledge, this study provides the first proof-of-concept that clonotypic analysis may be used as a tool to monitor the quality of vaccine-induced responses and modulate these toward “controller-like” responses.
HIV – Evolution
Liu, D., C. Wang, B. Hora, T. Zuo, N. Goonetilleke, M. K. P. Liu, M. Berrong, G. Ferrari, A. J. McMichael, T. Bhattacharya, A. S. Perelson and F. Gao (2017). “A strongly selected mutation in the HIV-1 genome is independent of T cell responses and neutralizing antibodies.” Retrovirology 14(1): 46.
BACKGROUND: Mutations rapidly accumulate in the HIV-1 genome after infection. Some of those mutations are selected by host immune responses and often cause viral fitness losses. This study is to investigate whether strongly selected mutations that are not associated with immune responses result in fitness losses. RESULTS: Strongly selected mutations were identified by analyzing 5′-half HIV-1 genome (gag/pol) sequences from longitudinal samples of subject CH0131. The K43R mutation in the gag gene was first detected at day 91 post screening and was fixed in the viral population at day 273 while the synonymous N323tc mutation was first detected at day 177 and fixed at day 670. No conventional or cryptic T cell responses were detected against either mutation sites by ELISpot analysis. However, when fitness costs of both mutations were measured by introducing each mutation into their cognate transmitted/founder (T/F) viral genome, the K43R mutation caused a significant fitness loss while the N323tc mutation had little impact on viral fitness. CONCLUSIONS: The rapid fixation, the lack of detectable immune responses and the significant fitness cost of the K43R mutation suggests that it was strongly selected by host factors other than T cell responses and neutralizing antibodies.
HIV - HLA
Chikata, T., H. Murakoshi, M. Koyanagi, K. Honda, H. Gatanaga, S. Oka and M. Takiguchi (2017). “Control of HIV-1 by an HLA-B*52:01-C*12:02 protective haplotype.” J Infect Dis.
HLA-B*52:01-C*12:02, which is found in approximately 20 % of all Japanese, is well known to be associated with ulcerative colitis and Takayasu arteritis. This haplotype is also known to be a protective one in HIV-1-infective individuals. Recent studies showed that HLA-B*52:01-restricted HIV-1-specific T cells suppress HIV-1 and that HLA-C*12:02 together with KIR2DL2 play an important role in NK cell-mediated control of HIV-1. However, the role of HLA-C*12:02-restricted CTLs in suppression of HIV-1 replication remains unknown. In the present study, we demonstrated that HLA-C*12:02-restricted CTLs specific for 2 immunodominant epitopes, Pol IY11 and Nef MY9, contributed to the suppression of HIV-1 replication in HIV-1-infected individuals. Further analysis demonstrated that these 2 HLA-C*12:02-restricted CTLs together with 4 HLA-B*52:01-restricted ones effectively suppressed HIV-1 in individuals having the HLA-B*52:01-C*12:02 haplotype. Thus, both HLA-C*12:02 and HLA-B*52:01 alleles contribute to HIV-1 suppression via both HIV-1-specific CTLs and NK cells in individuals having this haplotype.
Zhang, X. H., X. D. Lian, Z. X. Dai, H. Y. Zheng, X. Chen and Y. T. Zheng (2017). “alpha3-Deletion Isoform of HLA-A11 Modulates Cytotoxicity of NK Cells: Correlations with HIV-1 Infection of Cells.” J Immunol 199(6): 2030-2042.
Alternative splicing occurs frequently in many genes, especially those involved in immunity. Unfortunately, the functions of many alternatively spliced molecules from immunologically relevant genes remain unknown. Classical HLA-I molecules are expressed on almost all nucleated cells and play a pivotal role in both innate and adaptive immunity. Although splice variants of HLA-I genes have been reported, the details of their functions have not been reported. In the current study, we determined the characteristics, expression, and function of a novel splice variant of HLA-A11 named HLA-A11svE4 HLA-A11svE4 is located on the cell surface without beta2-microglobulin (beta2m). Additionally, HLA-A11svE4 forms homodimers as well as heterodimers with HLA-A open conformers, instead of combining with beta2m. Moreover, HLA-A11svE4 inhibits the activation of NK cells to protect target cells. Compared with beta2m and HLA-A11, the heterodimer of HLA-A11svE4 and HLA-A11 protected target cells from lysis by NK cells more effectively. Furthermore, HLA-AsvE4 expression was upregulated by HIV-1 in vivo and by HSV, CMV, and hepatitis B virus in vitro. In addition, our findings indicated that HLA-A11svE4 molecules were functional in activating CD8+ T cells through Ag presentation. Taken together, these results suggested that HLA-A11svE4 can homodimerize and form a novel heterodimeric complex with HLA-A11 open conformers. Furthermore, the data are consistent with HLA-A11svE4 playing a role in the immune escape of HIV-1.
HIV – Immune Activation
Abana, C. O., M. A. Pilkinton, S. Gaudieri, A. Chopra, W. J. McDonnell, C. Wanjalla, L. Barnett, R. Gangula, C. Hager, D. K. Jung, B. G. Engelhardt, M. H. Jagasia, P. Klenerman, E. J. Phillips, D. M. Koelle, S. A. Kalams and S. A. Mallal (2017). “Cytomegalovirus (CMV) Epitope-Specific CD4+ T Cells Are Inflated in HIV+ CMV+ Subjects.” J Immunol.
Select CMV epitopes drive life-long CD8+ T cell memory inflation, but the extent of CD4 memory inflation is poorly studied. CD4+ T cells specific for human CMV (HCMV) are elevated in HIV+ HCMV+ subjects. To determine whether HCMV epitope-specific CD4+ T cell memory inflation occurs during HIV infection, we used HLA-DR7 (DRB1*07:01) tetramers loaded with the glycoprotein B DYSNTHSTRYV (DYS) epitope to characterize circulating CD4+ T cells in coinfected HLA-DR7+ long-term nonprogressor HIV subjects with undetectable HCMV plasma viremia. DYS-specific CD4+ T cells were inflated among these HIV+ subjects compared with those from an HIV- HCMV+ HLA-DR7+ cohort or with HLA-DR7-restricted CD4+ T cells from the HIV-coinfected cohort that were specific for epitopes of HCMV phosphoprotein-65, tetanus toxoid precursor, EBV nuclear Ag 2, or HIV gag protein. Inflated DYS-specific CD4+ T cells consisted of effector memory or effector memory-RA+ subsets with restricted TCRbeta usage and nearly monoclonal CDR3 containing novel conserved amino acids. Expression of this near-monoclonal TCR in a Jurkat cell-transfection system validated fine DYS specificity. Inflated cells were polyfunctional, not senescent, and displayed high ex vivo levels of granzyme B, CX3CR1, CD38, or HLA-DR but less often coexpressed CD38+ and HLA-DR+ The inflation mechanism did not involve apoptosis suppression, increased proliferation, or HIV gag cross-reactivity. Instead, the findings suggest that intermittent or chronic expression of epitopes, such as DYS, drive inflation of activated CD4+ T cells that home to endothelial cells and have the potential to mediate cytotoxicity and vascular disease.
Leitman, E. M., C. F. Thobakgale, E. Adland, M. A. Ansari, J. Raghwani, A. J. Prendergast, G. Tudor-Williams, P. Kiepiela, J. Hemelaar, J. Brener, M. H. Tsai, M. Mori, L. Riddell, G. Luzzi, P. Jooste, T. Ndung’u, B. D. Walker, O. G. Pybus, P. Kellam, V. Naranbhai, P. C. Matthews, A. Gall and P. J. R. Goulder (2017). “Role of HIV-specific CD8+ T cells in pediatric HIV cure strategies after widespread early viral escape.” J Exp Med.
Recent studies have suggested greater HIV cure potential among infected children than adults. A major obstacle to HIV eradication in adults is that the viral reservoir is largely comprised of HIV-specific cytotoxic T lymphocyte (CTL) escape variants. We here evaluate the potential for CTL in HIV-infected slow-progressor children to play an effective role in “shock-and-kill” cure strategies. Two distinct subgroups of children were identified on the basis of viral load. Unexpectedly, in both groups, as in adults, HIV-specific CTL drove the selection of escape variants across a range of epitopes within the first weeks of infection. However, in HIV-infected children, but not adults, de novo autologous variant-specific CTL responses were generated, enabling the pediatric immune system to “corner” the virus. Thus, even when escape variants are selected in early infection, the capacity in children to generate variant-specific anti-HIV CTL responses maintains the potential for CTL to contribute to effective shock-and-kill cure strategies in pediatric HIV infection.
Li, H., M. Nykoluk, L. Li, L. R. Liu, R. W. Omange, G. Soule, L. T. Schroeder, N. Toledo, M. A. Kashem, J. F. Correia-Pinto, B. Liang, N. Schultz-Darken, M. J. Alonso, J. B. Whitney, F. A. Plummer and M. Luo (2017). “Natural and cross-inducible anti-SIV antibodies in Mauritian cynomolgus macaques.” PLoS One 12(10): e0186079.
Cynomolgus macaques are an increasingly important nonhuman primate model for HIV vaccine research. SIV-free animals without pre-existing anti-SIV immune responses are generally needed to evaluate the effect of vaccine-induced immune responses against the vaccine epitopes. Here, in order to select such animals for vaccine studies, we screened 108 naive female Mauritian cynomolgus macaques for natural (baseline) antibodies to SIV antigens using a Bio-Plex multiplex system. The antigens included twelve 20mer peptides overlapping the twelve SIV protease cleavage sites (-10/+10), respectively (PCS peptides), and three non-PCS Gag or Env peptides. Natural antibodies to SIV antigens were detected in subsets of monkeys. The antibody reactivity to SIV was further confirmed by Western blot using purified recombinant SIV Gag and Env proteins. As expected, the immunization of monkeys with PCS antigens elicited anti-PCS antibodies. However, unexpectedly, antibodies to non-PCS peptides were also induced, as shown by both Bio-Plex and Western blot analyses, while the non-PCS peptides do not share sequence homology with PCS peptides. The presence of natural and vaccine cross-inducible SIV antibodies in Mauritian cynomolgus macaques should be considered in animal selection, experimental design and result interpretation, for their best use in HIV vaccine research.
Nahabedian, J., A. Sharma, M. E. Kaczmarek, G. K. Wilkerson, S. L. Sawyer and J. Overbaugh (2017). “Owl monkey CCR5 reveals synergism between CD4 and CCR5 in HIV-1 entry.” Virology 512: 180-186.
Studying HIV-1 replication in the presence of functionally related proteins from different species has helped define host determinants of HIV-1 infection. Humans and owl monkeys, but not macaques, encode a CD4 receptor that permits entry of transmissible HIV-1 variants due to a single residue difference. However, little is known about whether divergent CCR5 receptor proteins act as determinants of host-range. Here we show that both owl monkey (Aotus vociferans) CD4 and CCR5 receptors are functional for the entry of transmitted HIV-1 when paired with human versions of the other receptor. By contrast, the owl monkey CD4/CCR5 pair is generally a suboptimal receptor combination, although there is virus-specific variation in infection with owl monkey receptors. Introduction of the human residues 15Y and 16T within a sulfation motif into owl monkey CCR5 resulted in a gain of function. These findings suggest there is cross-talk between CD4 and CCR5 involving the sulfation motif.
Root-Bernstein, R. (2017). “Human Immunodeficiency Virus Proteins Mimic Human T Cell Receptors Inducing Cross-Reactive Antibodies.” Int J Mol Sci 18(10).
Human immunodeficiency virus (HIV) hides from the immune system in part by mimicking host antigens, including human leukocyte antigens. It is demonstrated here that HIV also mimics the V-beta-D-J-beta of approximately seventy percent of about 600 randomly selected human T cell receptors (TCR). This degree of mimicry is greater than any other human pathogen, commensal or symbiotic organism studied. These data suggest that HIV may be evolving into a commensal organism just as simian immunodeficiency virus has done in some types of monkeys. The gp120 envelope protein, Nef protein and Pol protein are particularly similar to host TCR, camouflaging HIV from the immune system and creating serious barriers to the development of safe HIV vaccines. One consequence of HIV mimicry of host TCR is that antibodies against HIV proteins have a significant probability of recognizing the corresponding TCR as antigenic targets, explaining the widespread observation of lymphocytotoxic autoantibodies in acquired immunodeficiency syndrome (AIDS). Quantitative enzyme-linked immunoadsorption assays (ELISA) demonstrated that every HIV antibody tested recognized at least one of twelve TCR, and as many as seven, with a binding constant in the 10-8 to 10-9 m range. HIV immunity also affects microbiome tolerance in ways that correlate with susceptibility to specific opportunistic infections.
HIV – Neutralizing Antibodies
Cohen, M. S. and L. Corey (2017). “Broadly neutralizing antibodies to prevent HIV-1.” Science 358(6359): 46-47.
Advances in technology—especially single-cell antibody cloning techniques—have led to the isolation and characterization of antibodies from people with HIV infection that can neutralize many variants (1). These are referred to as broadly neutralizing antibodies (bnAbs). Such antibodies can be detected in about 25% of persons with untreated HIV-1 infection (2), reflecting a host immune response to unremitting viral replication, generation of large numbers of viral variants, and shifting antigen exposure (3). Although bnAbs may exert some selective pressure as they develop, they generally do not reduce viral burden, improve health, or slow the progression of disease (4). However, they offer considerable opportunities for treatment and prevention of HIV-1 infection in others. At this time, hundreds of bnAbs have been identified; those that have attracted the most attention are bnAbs with the greatest breadth, neutralizing the largest number of HIV-1 strains, including those traditionally most neutralization resistant (1, 5); or bnAbs that have the greatest potency, requiring the smallest concentration to neutralize resistant strains of HIV-1 (5–7). A study by Xu et al. (5) on page 85 of this issue and by Julg et al.(7) in Science Translational Medicine illustrate advances in the potential use of bnAbs to prevent HIV-1 infection.
Mayr, L. M., T. Decoville, S. Schmidt, G. Laumond, J. Klingler, C. Ducloy, S. Bahram, S. Zolla-Pazner and C. Moog (2017). “Non-neutralizing Antibodies Targeting the V1V2 Domain of HIV Exhibit Strong Antibody-Dependent Cell-mediated Cytotoxic Activity.” Sci Rep 7(1): 12655.
The development of an effective vaccine against HIV-1 has proven to be challenging. Broadly neutralizing antibodies (bNAbs), whilst exhibiting neutralization breadth and potency, are elicited only in a small subset of infected individuals and have yet to be induced by vaccination. Case-control studies of RV144 identified an inverse correlation of HIV-1 infection risk with antibodies (Abs) to the V1V2 region of gp120 with high antibody-dependent cellular cytotoxicity (ADCC) activity. The neutralizing activity of Abs was not found to contribute to this protective outcome. Using primary effector and target cells and primary virus isolates, we studied the ADCC profile of different monoclonal Abs targeting the V1V2 loop of gp120 that had low or no neutralizing activity. We compared their ADCC activity to some bNAbs targeting different regions of gp120. We found that mAbs targeting the V1V2 domain induce up to 60% NK cell mediated lysis of HIV-1 infected PBMCs in a physiologically relevant ADCC model, highlighting the interest in inducing such Abs in future HIV vaccine trials. Our data also suggest that in addition to neutralization, lysis of infected cells by Abs can effectively participate in HIV protection, as suggested by the RV144 immune correlate analysis.
Voss, J. E., R. Andrabi, L. E. McCoy, N. de Val, R. P. Fuller, T. Messmer, C. Y. Su, D. Sok, S. N. Khan, F. Garces, L. K. Pritchard, R. T. Wyatt, A. B. Ward, M. Crispin, I. A. Wilson and D. R. Burton (2017). “Elicitation of Neutralizing Antibodies Targeting the V2 Apex of the HIV Envelope Trimer in a Wild-Type Animal Model.” Cell Rep 21(1): 222-235.
Recent efforts toward HIV vaccine development include the design of immunogens that can engage B cell receptors with the potential to affinity mature into broadly neutralizing antibodies (bnAbs). V2-apex bnAbs, which bind a protein-glycan region on HIV envelope glycoprotein (Env) trimer, are among the most broad and potent described. We show here that a rare “glycan hole” at the V2 apex is enriched in HIV isolates neutralized by inferred precursors of prototype V2-apex bnAbs. To investigate whether this feature could focus neutralizing responses onto the apex bnAb region, we immunized wild-type rabbits with soluble trimers adapted from these Envs. Potent autologous tier 2 neutralizing responses targeting basic residues in strand C of the V2 region, which forms the core epitope for V2-apex bnAbs, were observed. Neutralizing monoclonal antibodies (mAbs) derived from these animals display features promising for subsequent broadening of the response.
Sun, Y., Y. Qiao, Y. Zhu, H. Chong and Y. He (2017). “Identification of a novel HIV-1-neutralizing antibody from a CRF07_BC-infected Chinese donor.” Oncotarget 8(38): 63047-63063.
The identification of human monoclonal antibodies (mAbs) able to neutralize a broad spectrum of primary HIV-1 isolates is highly important for understanding the immune response of HIV-1 infection and developing vaccines and therapeutics. In this study, we isolated a novel human mAb termed Y498 from a phage display antibody library constructed with the PBMC samples of a CRF07_BC-infected Chinese donor whose sera exhibited broadly neutralizing activity. Y498 cross-reacted with diverse Env antigens and neutralized 30% of 70 tested HIV-1 isolates. It efficiently blocked the binding of soluble CD4 to gp120 and competed with the CD4-binding site (CD4bs)-specific mAbs. By combining molecular docking and site-directed mutagenesis, the epitope of Y498 was characterized to contain three antigenic sites on gp120, including the CD4 binding loop in C3, the beta23 in C4 and the beta24-alpha5 in C5, which overlap the binding sites of CD4 and CD4bs-directed mAbs (b12, VRC01, A16). Therefore, Y498 is a novel neutralizing human mAb targeting a conformation-dependent CD4bs-based epitope, and its isolation and characterization could provide helpful information for elucidating human immune response to HIV-1 infection and designing effective vaccines and immunotherapeutics.
HIV – PrEP in Africa
Bekker, L. G., S. Roux, E. Sebastien, N. Yola, K. R. Amico, J. P. Hughes, M. A. Marzinke, C. W. Hendrix, P. L. Anderson, V. Elharrar, M. Stirratt, J. F. Rooney, E. Piwowar-Manning, S. H. Eshleman, L. McKinstry, M. Li, B. J. Dye, R. M. Grant and H. s. team (2017). “Daily and non-daily pre-exposure prophylaxis in African women (HPTN 067/ADAPT Cape Town Trial): a randomised, open-label, phase 2 trial.” Lancet HIV.
BACKGROUND: The relative feasibility and acceptability of daily versus non-daily dosing of oral HIV pre-exposure prophylaxis (PrEP) among women are unknown. We aimed to investigate the feasibility of non-daily PrEP regimens in adult women. METHODS: We did a randomised, open-label, phase 2 clinical trial (HPTN 067/ADAPT) of oral PrEP with emtricitabine plus tenofovir disoproxil fumarate at a research centre in Cape Town, South Africa. Participants were adult women (age >/=18 years) who received directly observed dosing once a week for 5 weeks followed by random assignment (1:1:1) at week 6 to one of three unblinded PrEP regimens for self-administered dosing over 24 weeks: daily; time-driven (twice a week plus a post-sex dose); or event-driven (one tablet both before and after sex). Primary outcomes were PrEP coverage (at least one dose within the 4 days before sex and one dose within 24 h after sex), pills needed or used to achieve regimen-specific adherence and coverage, and symptoms and side-effects. All analyses were by intention to treat. This trial is registered with ClinicalTrials.gov, number NCT01327651; the trial is completed and this report presents the final analysis. FINDINGS: Between Sept 12, 2011, and Oct 3, 2012, 191 women were enrolled to the trial. 178 (93%) completed directly observed dosing and were randomly assigned one of the three PrEP regimens for the self-administered phase: 59 were allocated the daily regimen, 59 the time-driven regimen, and 60 the event-driven regimen. Median age of women was 26 years (IQR 21-37; range 18-52). In women allocated the daily regimen, 1459 (75%) of 1952 sex events were covered by PrEP, compared with 599 (56%) of 1074 sex events among those assigned the time-driven regimen (odds ratio [OR] 2.35, 95% CI 1.43-3.83; p=0.0007) and 798 (52%) of 1542 sex events among those allotted the event-driven regimen (2.76, 1.68-4.53; p<0.0001). Fewer pills were needed for complete adherence in women allocated non-daily regimens (vs daily regimen, relative mean 2.53 [95% CI 2.39-2.69] for the time-driven regimen and 4.16 [3.59-4.82] for the event-driven regimen; p<0.0001). Side-effects were uncommon. Eight HIV seroconversions occurred overall, with four documented during the self-administered phase (two with the time-driven regimen and two with the event-driven regimen). Adherence to the assigned regimen was 75% (7283 of 9652 doses taken) for women allocated the daily regimen compared with 65% for those assigned the time-driven regimen (2367 of 3616 doses taken; p=0.0028) and 53% for those allotted the event-driven regimen (1161 of 2203 doses taken; p<0.0001). When sex was reported in the previous week, PrEP drugs were detected (above the lower limits of quantification) more frequently in women assigned the daily regimen (73 [68%] of 107 samples) than in those allocated the time-driven regimen (42 [58%] of 72 samples) and the event-driven regimen (41 [41%] of 99 samples). INTERPRETATION: Daily PrEP dosing resulted in higher coverage of sex events, increased adherence to the regimen, and augmented drug concentrations than did either time-driven or event-driven dosing. These findings support recommendations for daily use of PrEP with oral emtricitabine plus tenofovir disoproxil fumarate in women. FUNDING: HIV Prevention Trials Network.
Pyra, M., J. E. Haberer, R. Heffron, L. Kidoguchi, E. R. Brown, E. A. Bukusi, S. Asiimwe, C. Celum, E. Katabira, N. R. Mugo and J. M. Baeten (2017). “PrEP Use During Periods of HIV Risk Among East African Women in Serodiscordant Relationships.” J Acquir Immune Defic Syndr.
BACKGROUND: PrEP is efficacious for African women at risk for HIV, but data on adherence outside of clinical trials are sparse. We describe the persistence and execution of PrEP use among women participating in a large open-label PrEP demonstration project, particularly during periods of HIV risk. SETTING: & Methods: 310 HIV-uninfected women in HIV serodiscordant couples in Kenya and Uganda were offered and accepted PrEP. Electronic monitoring caps were used to measure daily PrEP adherence. Time on PrEP while at risk for HIV (when the HIV-infected partner was on ART <6 months) and weekly adherence while on PrEP were calculated and compared among older and younger (<25 years old) women. RESULTS: As defined above, women were at risk for HIV for an average of 361 days; 54% took PrEP during their entire risk period and 24% stopped but re-started PrEP during their risk period. While on PrEP, women took >/=6 doses/week for 78% of weeks (67% of weeks for women <25 years old, 80% of weeks for women >/=25 years [p<0.001]), and >/=4 doses for 88% of weeks (80% for those <25, 90% for those >/=25, [p<0.001]). Compared to historical, risk-matched controls, HIV incidence was reduced 93% (95% CI 77%-98%) for all women and 91% (95% CI 29%-99%) among women <25 years old. CONCLUSION: Women, including young women, in HIV-serodiscordant couples took PrEP successfully over sustained periods of risk. While young women had lower adherence than older women, they achieved strong protection, suggesting women can align PrEP use to periods of risk and imperfect adherence can still provide substantial benefit. This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial License 4.0 (CCBY-NC), where it is permissible to download, share, remix, transform, and buildup the work provided it is properly cited. The work cannot be used commercially without permission from the journal.
Bracq, L., M. Xie, M. Lambele, L. T. Vu, J. Matz, A. Schmitt, J. Delon, P. Zhou, C. Randriamampita, J. Bouchet and S. Benichou (2017). “T cell-macrophage fusion triggers multinucleated giant cell formation for HIV-1 spreading.” J Virol.
HIV-1-infected macrophages participate in virus dissemination and establishment of virus reservoirs in host tissues, but the mechanisms for virus cell-to-cell transfer to macrophages remain unknown. Here, we reveal the mechanisms for cell-to-cell transfer from infected T cells to macrophages and virus spreading between macrophages. We show that contacts between infected T lymphocytes and macrophages lead to cell fusion for fast and massive transfer of CCR5-tropic viruses to macrophages. Through the merge of viral material between T cells and macrophages, these newly formed lymphocyte/macrophage fused cells acquire the ability to fuse with neighboring non-infected macrophages. Together, these two-step envelope-dependent cell fusion processes lead to the formation of highly virus-productive multinucleated giant cells reminiscent of the infected multinucleated giant macrophages detected in HIV-1-infected patients and SIV-infected macaques. These mechanisms represent an original mode of virus transmission for viral spreading and a new model for the formation of macrophage virus reservoirs during infection. IMPORTANCE: We reveal a very efficient mechanism involved in cell-to-cell transfer from infected T cells to macrophages and subsequent virus spreading between macrophages by a two-step cell fusion process. Infected T cells first establish contacts and fuse with macrophage targets. The newly formed lymphocyte/macrophage fused cells then acquire the ability to fuse with surrounding uninfected macrophages leading to the formation of infected multinucleated giant cells that can survive for a long time as evidenced in vivo in lymphoid organs and the central nervous system. This route of infection may be a major determinant for virus dissemination and the formation of macrophage virus reservoirs in host tissues during HIV-1 infection.
McCurley, N. P., A. Domi, R. Basu, K. O. Saunders, C. C. LaBranche, D. C. Montefiori, B. F. Haynes and H. L. Robinson (2017). “HIV transmitted/founder vaccines elicit autologous tier 2 neutralizing antibodies for the CD4 binding site.” PLoS One 12(10): e0177863.
Here we report the construction, antigenicity and initial immunogenicity testing of DNA and modified vaccinia Ankara (MVA) vaccines expressing virus-like particles (VLPs) displaying sequential clade C Envelopes (Envs) that co-evolved with the elicitation of broadly neutralizing antibodies (bnAbs) to the CD4 binding site (CD4bs) in HIV-infected individual CH0505. The VLP-displayed Envs showed reactivity for conformational epitopes displayed on the receptor-binding form of Env. Two inoculations of the DNA-T/F vaccine, followed by 3 inoculations of the MVA-T/F vaccine and a final inoculation of the MVA-T/F plus a gp120-T/F protein vaccine elicited nAb to the T/F virus in 2 of 4 rhesus macaques (ID50 of ~175 and ~30). Neutralizing Ab plateaued at 100% neutralization and mapped to the CD4bs like the bnAbs elicited in CH0505. The nAb did not have breadth for other tier 2 viruses. Immunizations with T/F followed by directed-lineage vaccines, both with and without co-delivery of directed-lineage gp120 boosts, failed to elicit tier 2 neutralizing Ab for the CD4bs. Thus, pulsed exposures to DNA and MVA-expressed VLPs plus gp120 protein of a T/F Env can induce autologous tier 2 nAbs to the CD4bs.
Puller, V., R. Neher and J. Albert (2017). “Estimating time of HIV-1 infection from next-generation sequence diversity.” PLoS Comput Biol 13(10): e1005775.
Estimating the time since infection (TI) in newly diagnosed HIV-1 patients is challenging, but important to understand the epidemiology of the infection. Here we explore the utility of virus diversity estimated by next-generation sequencing (NGS) as novel biomarker by using a recent genome-wide longitudinal dataset obtained from 11 untreated HIV-1-infected patients with known dates of infection. The results were validated on a second dataset from 31 patients. Virus diversity increased linearly with time, particularly at 3rd codon positions, with little inter-patient variation. The precision of the TI estimate improved with increasing sequencing depth, showing that diversity in NGS data yields superior estimates to the number of ambiguous sites in Sanger sequences, which is one of the alternative biomarkers. The full advantage of deep NGS was utilized with continuous diversity measures such as average pairwise distance or site entropy, rather than the fraction of polymorphic sites. The precision depended on the genomic region and codon position and was highest when 3rd codon positions in the entire pol gene were used. For these data, TI estimates had a mean absolute error of around 1 year. The error increased only slightly from around 0.6 years at a TI of 6 months to around 1.1 years at 6 years. Our results show that virus diversity determined by NGS can be used to estimate time since HIV-1 infection many years after the infection, in contrast to most alternative biomarkers. We provide the regression coefficients as well as web tool for TI estimation.
Wykes, M. N. and S. R. Lewin (2017). “Immune checkpoint blockade in infectious diseases.” Nat Rev Immunol.
The upregulation of immune checkpoint molecules, such as programmed cell death protein 1 (PD1) and cytotoxic T lymphocyte antigen 4 (CTLA4), on immune cells occurs during acute infections, such as malaria, as well as during chronic persistent viral infections, including HIV and hepatitis B virus. These pathways are important for preventing immune-driven pathology but can also limit immune-mediated clearance of the infection. The recent success of immune checkpoint blockade in cancer therapy suggests that targeting these pathways would also be effective for preventing and treating a range of infectious diseases. Here, we review our current understanding of immune checkpoint pathways in the pathogenesis of infectious diseases and discuss the potential for therapeutically targeting these pathways in this setting.
Lisa van Sluijs, L., G. P. Pijlman and J. E. Kammenga (2017). “Why do Individuals Differ in Viral Susceptibility? A Story Told by Model Organisms.” Viruses 9(10).
Viral susceptibility and disease progression is determined by host genetic variation that underlies individual differences. Genetic polymorphisms that affect the phenotype upon infection have been well-studied for only a few viruses, such as HIV-1 and Hepatitis C virus. However, even for well-studied viruses the genetic basis of individual susceptibility differences remains elusive. Investigating the effect of causal polymorphisms in humans is complicated, because genetic methods to detect rare or small-effect polymorphisms are limited and genetic manipulation is not possible in human populations. Model organisms have proven a powerful experimental platform to identify and characterize polymorphisms that underlie natural variations in viral susceptibility using quantitative genetic tools. We summarize and compare the genetic tools available in three main model organisms, Mus musculus, Drosophila melanogaster, and Caenorhabditis elegans, and illustrate how these tools can be applied to detect polymorphisms that determine the viral susceptibility. Finally, we analyse how candidate polymorphisms from model organisms can be used to shed light on the underlying mechanism of individual variation. Insights in causal polymorphisms and mechanisms underlying individual differences in viral susceptibility in model organisms likely provide a better understanding in humans.
Vaccine Design Immunogenicity Efficacy
Suschak, J. J. and C. S. Schmaljohn (2018). “Future Approaches to DNA Vaccination Against Hemorrhagic Fever Viruses.” Methods Mol Biol 1604: 339-348.
To date, there is no protective vaccine for Ebola virus infection. Safety concerns have prevented the use of live-attenuated vaccines, and forced researchers to examine new vaccine formulations. DNA vaccination is an attractive method for inducing protective immunity to a variety of pathogens, but the low immunogenicity seen in larger animals and humans has hindered its usage. Various approaches have been used to improve the immunogenicity of DNA vaccines, but the most successful, and widespread, is electroporation. Of increasing interest is the use of molecular adjuvants to produce immunomodulatory signals that can both amplify and direct the immune response. When combined, these approaches have the possibility to push DNA vaccination into the forefront of medicine.
Smith, S. G., S. A. Harris, I. Satti, D. Bryan, K. B. Walker, H. M. Dockrell, H. McShane and M. M. Ho (2017). “Assay optimisation and technology transfer for multi-site immuno-monitoring in vaccine trials.” PLoS One 12(10): e0184391.
Cellular immunological assays are important tools for the monitoring of responses to T-cell-inducing vaccine candidates. As these bioassays are often technically complex and require considerable experience, careful technology transfer between laboratories is critical if high quality, reproducible data that allows comparison between sites, is to be generated. The aim of this study, funded by the European Union Framework Program 7-funded TRANSVAC project, was to optimise Standard Operating Procedures and the technology transfer process to maximise the reproducibility of three bioassays for interferon-gamma responses: enzyme-linked immunosorbent assay (ELISA), ex-vivo enzyme-linked immunospot and intracellular cytokine staining. We found that the initial variability in results generated across three different laboratories reduced following a combination of Standard Operating Procedure harmonisation and the undertaking of side-by-side training sessions in which assay operators performed each assay in the presence of an assay ‘lead’ operator. Mean inter-site coefficients of variance reduced following this training session when compared with the pre-training values, most notably for the ELISA assay. There was a trend for increased inter-site variability at lower response magnitudes for the ELISA and intracellular cytokine staining assays. In conclusion, we recommend that on-site operator training is an essential component of the assay technology transfer process and combined with harmonised Standard Operating Procedures will improve the quality, reproducibility and comparability of data produced across different laboratories. These data may be helpful in ongoing discussions of the potential risk/benefit of centralised immunological assay strategies for large clinical trials versus decentralised units.
Vaccine Design – Vectors
Barbieri, A., M. Panigada, E. Soprana, G. D. Mario, F. Gubinelli, V. Bernasconi, M. Recagni, I. Donatelli, M. R. Castrucci and A. G. Siccardi (2017). “Strategies to obtain multiple recombinant Modified Vaccinia Ankara vectors. Applications to influenza vaccines.” J Virol Methods.
As a vaccination vector, MVA has been widely investigated both in animal models and humans. The construction of recombinant MVA (rMVA) relies on homologous recombination between an acceptor virus and a donor plasmid in infected/transfected permissive cells. Our construction strategy “Red-to-Green gene swapping” – based on the exchange of two fluorescent markers within the flanking regions of MVA deletion DeltaIII, coupled to fluorescence activated cell sorting – is here extended to a second insertion site, within the flanking regions of MVA deletion DeltaVI. Exploiting this strategy, both double and triple rMVA were constructed, expressing as transgenes the influenza A proteins HA, NP, M1, and PB1. Upon validation of the harbored transgenes co-expression, double and triple recombinants rMVA(DeltaIII)-NP-P2A-M1 and rMVA(DeltaIII)-NP-P2A-M1-(DeltaVI)-PB1 were assayed for in vivo immunogenicity and protection against lethal challenge. In vivo responses were identical to those obtained with the reported combinations of single recombinants, supporting the feasibility and reliability of the present improvement and the extension of Red-to-Green gene swapping to insertion sites other than DeltaIII.
Kratzer, R. F. and F. Kreppel (2017). “Production, Purification, and Titration of First-Generation Adenovirus Vectors.” Methods Mol Biol1654: 377-388.
Vectors based on human adenovirus are highly efficient tools for transient genetic modifications of cells or tissues in vitro and in vivo. They can be utilized for gene addition strategies, knockdown strategies and as transfer vectors for designer nucleases and CRISPR/Cas. They are characterized by high genomic stability and can be produced to high titers. This chapter describes the method how to produce, purify and titrate adenovirus vectors based on human adenovirus type 5.