Broadly neutralizing antibodies and vaccine design against HIV-1 infection.

Remarkable progress has been achieved for prophylactic and therapeutic interventions against human immunodeficiency virus type I (HIV-1) through antiretroviral therapy. However, vaccine development has remained challenging. Recent discoveries in broadly neutralizing monoclonal antibodies (bNAbs) has led to the development of multiple novel vaccine approaches for inducing bNAbs-like antibody response. Structural and dynamic studies revealed several vulnerable sites and states of the HIV-1 envelop glycoprotein (Env) during infection. Our review aims to highlight these discoveries and rejuvenate our endeavor in HIV-1 vaccine design and development.

Isolation of single HIV-1 Envelope specific B cells and antibody cloning from immunized rhesus macaques.

Antibody cloning from single B cells is an essential tool for characterizing humoral immune responses and obtaining valuable therapeutic and analytical reagents. Antibody cloning from individuals with high serologic titers to HIV-1, Influenza, Malaria and ZIKV has led to new insights that inform vaccine design efforts. In contrast to humans and mice, less is known about antibody cloning from single B cells in macaques. Here, we describe a protocol to identify and purify single antigen-specific macaque B cells, and subsequently clone and produce macaque monoclonal antibodies. The sorting strategy requires the use of a combination of fluorochrome labeled antigens and omission of anti-IgG antibodies that can interfere with antigen binding and vice versa. Optimized methods for macaque antibody gene amplification, DNA preparation for antibody production and antibody screening by ELISA are also presented.

Designing Chimeric Virus-like Particle-based Vaccines for Human Papillomavirus and HIV: Lessons Learned.

Virus-like particles (VLPs) are a type of subunit vaccine which resembles viruses but do not contain any genetic material so that they are not infectious. VLPs maintain the same antigenic conformation to the original virus, and they could be a better vaccine candidate than live-attenuated and inactivated vaccines. In addition, compared to other subunit vaccines such as soluble protein, VLPs can stimulate both innate and adaptive immune responses effectively and safely against several pathogens by the closer morphology to its native virus. They have already been licensed as vaccines against Hepatitis B virus, human papillomavirus (HPV), and several veterinary diseases. Moreover, it has been investigated to prevent other viral infections including HIV. While HIV VLP-based vaccines have been studied over 35 years, none of them has been successful enough to reach even Phase III clinical trials. In this review, we summarize: (i) general features of VLPs; (ii) epidemiological data and current status of vaccine research and development on HPV and HIV; and (iii) previous studies held on HPV VLPs, HIV VLPs, and chimeric HPV/HIV VLPs including production methods and different animal immunization assays. Furthermore, we review present state of human clinical trials with VLPs and consider the potential to develop a successful preventive HIV vaccine using HPV VLP models. Finally, we discuss the benefits, limitations, and challenges of developing chimeric VLP-based HPV/HIV vaccines with recent findings, critical issues to improve VLP-based vaccines, and hot topics for the next 5 years to join the global effort to fight against these two pathogens.

Computational Design of Epitope-Enriched HIV-1 Gag Antigens with Preserved Structure and Function for Induction of Broad CD8+ T Cell Responses.

The partially protective phenotype observed in HIV-infected long-term-non-progressors is often associated with certain HLA alleles, thus indicating that cytotoxic T lymphocyte (CTL) responses play a crucial role in combating virus replication. However, both the vast variability of HIV and the HLA diversity impose a challenge on elicitation of broad and effective CTL responses. Therefore, we conceived an algorithm for the enrichment of CD8+ T cell epitopes in HIV's Gag protein, respecting functional preservation to enable cross-presentation. Experimentally identified epitopes were compared to a Gag reference sequence. Amino-acid-substitutions (AAS) were assessed for their impact on Gag's budding-function using a trained classifier that considers structural models and sequence conservation. Experimental assessment of Gag-variants harboring selected AAS demonstrated an apparent classifier-precision of 100%. Compatible epitopes were assigned an immunological score that incorporates features such as conservation or HLA-association in a user-defined weighted manner. Using a genetic algorithm, the epitopes were incorporated in an iterative manner into novel T-cell-epitope-enriched Gag sequences (TeeGag). Computational evaluation showed that these antigen candidates harbor a higher fraction of epitopes with higher score as compared to natural Gag isolates and other artificial antigen designs. Thus, these designer sequences qualify as next-generation antigen candidates for induction of broader CTL responses.

Harnessing T Follicular Helper Cell Responses for HIV Vaccine Development.

Passive administration of broadly neutralizing antibodies (bNAbs) capable of recognizing a broad range of viral strains to non-human primates has led to protection from infection with chimeric SIV/HIV virus (SHIV). This data suggests that generating protective antibody responses could be an effective strategy for an HIV vaccine. However, classic vaccine approaches have failed so far to induce such protective antibodies in HIV vaccine trials. HIV-specific bNAbs identified in natural infection show high levels of somatic hypermutations, demonstrating that they underwent extensive affinity maturation. It is likely that to gain ability to recognize diverse viral strains, vaccine-induced humoral responses will also require complex, iterative maturation. T follicular helper cells (Tfh) are a specialized CD4+ T cell subset that provides help to B cells in the germinal center for the generation of high-affinity and long-lasting humoral responses. It is therefore probable that the quality and quantity of Tfh responses upon vaccination will impact development of bNAbs. Here, we review studies that advanced our understanding of Tfh differentiation, function and regulation. We discuss correlates of Tfh responses and bNAb development in natural HIV infection. Finally, we highlight recent strategies to optimize Tfh responses upon vaccination and their impact on prophylactic HIV vaccine research.

Natural Immunity to HIV: A Template for Vaccine Strategies.

Africa accounts for the majority of global human immunodeficiency virus (HIV) infections, most of which affect women through heterosexual intercourse. Currently, there is no cure for HIV and the development of vaccines and microbicides remains the best solution to eradicate the pandemic. We and others have identified HIV highly-exposed seronegative (HESN) individuals among African female commercial sex workers (CSWs). Analyses of genital samples from HESNs have demonstrated potent innate and anti-inflammatory conditions, HIV-specific CD4⁺ and CD8⁺ T-cells as well as immunoglobulins (Igs), and increased regulatory cell populations, all of which support a delicate balance between strength and control against HIV intrusion. Moreover, we have recently shown that frequencies of innate marginal zone (MZ) B-cells are decreased in the blood of HESNs when compared to HIV-uninfected non-CSW women, suggesting their recruitment to peripheral sites. This coincides with the fact that levels of B lymphocyte stimulator (BLyS/BAFF), known to shape the MZ pool and whose overexpression leads to MZ deregulation in HIV-infected progressors, are significantly lower in the blood of HESNs when compared to both HIV-infected CSWs and HIV-uninfected non-CSW women. Interestingly, MZ B-cells can bind HIV gp120 and produce specific IgG and IgA, and have a propensity for B regulatory potential, which could help both the fight against HIV and maintenance of low inflammatory conditions in HESNs. HESN individuals provide an exceptional opportunity to identify important clues for the development of protective devices, and efforts should aim at soliciting immune responses observed in the context of their natural immunity to HIV.

The Hard Way towards an Antibody-Based HIV-1 Env Vaccine: Lessons from Other Viruses.

Although effective antibody-based vaccines have been developed against multiple viruses, such approaches have so far failed for the human immunodeficiency virus type 1 (HIV-1). Despite the success of anti-retroviral therapy (ART) that has turned HIV-1 infection into a chronic disease and has reduced the number of new infections worldwide, a vaccine against HIV-1 is still urgently needed. We discuss here the major reasons for the failure of "classical" vaccine approaches, which are mostly due to the biological properties of the virus itself. HIV-1 has developed multiple mechanisms of immune escape, which also account for vaccine failure. So far, no vaccine candidate has been able to induce broadly neutralizing antibodies (bnAbs) against primary patient viruses from different clades. However, such antibodies were identified in a subset of patients during chronic infection and were shown to protect from infection in animal models and to reduce viremia in first clinical trials. Their detailed characterization has guided structure-based reverse vaccinology approaches to design better HIV-1 envelope (Env) immunogens. Furthermore, conserved Env epitopes have been identified, which are promising candidates in view of clinical applications. Together with new vector-based technologies, considerable progress has been achieved in recent years towards the development of an effective antibody-based HIV-1 vaccine.

Multi-Envelope HIV-1 Vaccine Development: Two Targeted Immune Pathways, One Desired Protective Outcome.

In 2016, there were more than 30 million individuals living with HIV-1, ∼1.8 million new HIV-1 infections, and ∼1 million HIV-1-related deaths according to UNAIDS ( unaids.org ). Hence, a preventive HIV-1 vaccine remains a global priority. The variant envelopes of HIV-1 present a significant obstacle to vaccine development and the vaccine field has realized that immunization with a single HIV-1 envelope protein will not be sufficient to generate broadly neutralizing antibodies. Here we describe two nonmutually exclusive, targeted pathways with which a multi-envelope HIV-1 vaccine may generate protective immune responses against variant HIV-1. Pathways include (i) the induction of a polyclonal immune response, comprising a plethora of antibodies with subset-reactive and cross-reactive specificities, together able to neutralize diverse HIV-1 (termed Poly-nAb in this report) and (ii) the induction of one or a few monoclonal antibodies, each with a broadly neutralizing specificity (bnAb). With each pathway in mind, we describe challenges and strategies that may ultimately support HIV-1 vaccine success.

Immunization with Clinical HIV-1 Env Proteins Induces Broad Antibody Dependent Cellular Cytotoxicity-Mediating Antibodies in a Rabbit Vaccination Model.

The induction of both neutralizing antibodies and non-neutralizing antibodies with effector functions, for example, 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 peripheral blood mononuclear cells 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.

Construction and Production of HIV-VLP Harboring MPER-V3 for Potential Vaccine Study.

BACKGROUND: Vaccine against HIV-1 is not currently available. In present, Virus like particles (VLPs) as effective strategy was used in several vaccine developing. Two conserved sequences; V3 loop of gp120 and the membrane-proximal external region (MPER) of gp41 are dominant sites for vaccine studies. OBJECTIVE: In this study, we used fusion gene of MPER and V3 to product recombinant VLPs and introduced a novel retroviral VLPs harboring high copy of MPER-V3 for HIV-1 vaccine design. METHODS: The pEGFP-N1 plasmid harboring MPER-V3 sequence with Vpr linker was constructed. To produce virus-like particles, HEK 293T cells were co-transfected with the recombinant plasmid, pSPAX-2, pMD2-G and pWPXLd plasmids, evaluated by AFM and SEM microscopy and quantified using P24 end-point ELISA assay. RESULTS: Time-course quantification of p24 protein as the characteristics of viral production evidenced for the efficient secretion of virus-like structures (up to 120 ng/ml) to the culture supernatant of transfected cells. Examination of the centrifuge-concentrated VLPs by AFM and SEM microscope, also illustrated particles with spherical morphologies and diameters of around 150 nm that had similar sizes to HIV virions. CONCLUSION: These data indicated the production of HIV-1 virus-like particles harboring high copy of MPER-V3 that maintained their antigenic structure. These VLPs represented a good implication as a potential vaccine candidate and this guarantees the further investigations towards the assessment of its immunogenicity.

Hope for HIV control in southern Africa: The continued quest for a vaccine.

In a Perspective, Linda-Gail Bekker and Glenda Gray discuss HIV vaccine development.

Expression, purification, and renaturation of a recombinant peptide-based HIV vaccine in Escherichia coli.

To design an epitope-based vaccine for Human immunodeficiency virus (HIV), we previously predicted 20 potential HIV epitopes using bioinformatics approaches. The combination of these 20 epitopes has a theoretical coverage of 98.1% of the population for both the prevalent HIV genotypes and Chinese human leukocyte antigen DR types. To test the immunogenicity of this vaccine in vivo, a corresponding antigen needs to be prepared. To this end, we constructed a recombinant plasmid containing DNA encoding the epitopes and GPGPG spacers and a 6-His tag for verification of protein expression and ease of purification, and then transformed Escherichia coli cells with the plasmid. After IPTG induction, the recombinant protein was expressed in the form of mainly inclusion bodies. To stabilize the structure of denatured inclusion bodies for efficient purification and renaturation in vitro, we transferred the dissolved inclusion bodies from 7 mol/L guanidine hydrochloride to 8 mol/L urea. Under denaturing conditions, the vaccine protein was purified by a 3-step process including ion-exchange chromatography and affinity column, and then renatured by stepwise dialysis. Together, the above described procedures generated 43 mg of vaccine protein per litre of fermentation medium, and the final product reached approximately 95% purity. The purified protein was capable of eliciting antigen-specific T-cell responses in immunized mice.

Natural infection as a blueprint for rational HIV vaccine design.

So far, the development of a human immunodeficiency virus (HIV) vaccine has been unsuccessful. However, recent progress in the field of broadly neutralizing antibodies (bNAbs) has reinvigorated the search for an HIV vaccine. bNAbs develop in a minority of HIV infected individuals and passive transfer of these bNAbs to non-human primates provides protection from HIV infection. Studies in a number of HIV infected individuals on bNAb maturation alongside viral evolution and escape have shed light on the features important for bNAb elicitation. Here we review the observations from these studies, and how they influence the rational design of HIV vaccines.

Modeling HIV vaccine trials of the future.

PURPOSE OF REVIEW: Models of implementation of known-effective interventions for HIV prevention indicate that an efficacious vaccine to prevent HIV infection would be critical for controlling the HIV pandemic. Key issues in the design of future HIV vaccine trials are: first, how to develop reliable immunological correlates of vaccine efficacy, second, how to down-select candidate vaccine regimens into efficacy trials, and third, how to learn about vaccine efficacy in the context of the evolving HIV prevention landscape. RECENT FINDINGS: Whereas in the past phase-I/-II HIV vaccine trials have addressed the first and second points using a small set of immunological assays and readouts, recently they have used a battery of assays with highly multivariate readouts. In addition, systems vaccinology studies of other pathogens measuring PBMC transcriptomics and other immunological features pre- and postfirst vaccination are demonstrating value, for example, providing discoveries that preimmunization and early postimmunization cell population markers can predict the influenza-specific antibody titer that is a correlate of vaccine protection. The HIV prevention landscape continues to evolve, and the design and analysis of vaccine trials is evolving alongside, to accommodate increasingly dynamic and regional standards of HIV prevention. SUMMARY: Development of interpretable and robust functional assays, in addition to the associated bioinformatics and statistical analytic tools, is needed to improve the assessment of correlates of protection in efficacy trials and the down-selection of candidate vaccine regimens into efficacy trials. Moreover, high-priority trials should integrate systems vaccinology, including the analysis of prevaccination and early postvaccination markers.

Novel approaches in preclinical HIV vaccine research.

PURPOSE OF REVIEW: The purpose is to review recent novel approaches in HIV vaccine research and development being undertaken in the preclinical and early clinical space, as well as related and novel nonvaccine approaches such as genetic delivery of broadly neutralizing antibodies for protection from HIV infection and AIDS. RECENT FINDINGS: We review novel HIV envelope immunogen design, including native trimer and germline targeting approaches as well as genetic delivery of broadly neutralizing antibodies and replicating vector vaccinesSUMMARY: Despite 30+ years of research and development, and billions of dollars spent, a well tolerated and effective HIV vaccine remains a public health priority for any chance of ending the AIDS pandemic. It has become very clear that significant investments in novel technologies, innovation, and multidisciplinary science will be necessary to accelerate progress.

Approaches to the induction of HIV broadly neutralizing antibodies.

PURPOSE OF REVIEW: A vaccine that elicits antibody responses that can neutralize the diversity of HIV clades has not yet been achieved, and is a major focus of HIV vaccine research. Here, we provide an update on the barriers to eliciting such antibodies, and how advances in immunogen design may circumvent these roadblocks, focusing on data published in the last year. RECENT FINDINGS: Studies of how broadly neutralizing antibodies (bNAbs) develop in HIV-infected donors continue to produce key insights, suggesting that for some viral targets there are common pathways to developing breadth. Germline-targeting strategies, that aim to recruit rare precursors of bNAbs, have shown promise in immunogenicity studies, and structural biology has led to advances in immunogen design. Mapping of strain-specific tier 2 vaccine responses has highlighted the challenges that remain in driving antibodies toward breadth. SUMMARY: Elucidation of the HIV envelope structure, together with an understanding of how bNAbs emerge in vivo has guided the design of new immunogens and vaccine strategies that show promise for eliciting protective antibodies.