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Epitopes with evidence of HLA-II-associated adaptation induce poorly immunogenic CD4 + T-cell responses in HIV-positive (HIV + ) individuals. Many such escaped CD4 + T-cell epitopes are encoded by HIV-1 vaccines being evaluated in clinical trials. Here, we assessed whether this viral adaptation adversely impacts CD4 + T-cell responses following HIV-1 vaccination, thereby representing escaped epitopes. When evaluated in separate peptide pools, vaccine-encoded adapted epitopes (AE) induced CD4 + T-cell responses less frequently than nonadapted epitopes (NAE). We also demonstrated that in a polyvalent vaccine, where both forms of the same epitope were encoded, AE were less immunogenic. NAE-specific CD4 + T cells had increased, albeit low, levels of interferon gamma (IFN-γ) cytokine production. Single-cell transcriptomic analyses showed that NAE-specific CD4 + T cells expressed interferon-related genes, while AE-specific CD4 + T cells resembled a Th2 phenotype. Importantly, the magnitude of NAE-specific CD4 + T-cell responses, but not that of AE-specific responses, was found to positively correlate with Env-specific antibodies in a vaccine efficacy trial. Together, these findings show that HLA-II-associated viral adaptation reduces CD4 + T-cell responses in HIV-1 vaccine recipients and suggest that vaccines encoding a significant number of AE may not provide optimal B-cell help for HIV-specific antibody production. IMPORTANCE Despite decades of research, an effective HIV-1 vaccine remains elusive. Vaccine strategies leading to the generation of broadly neutralizing antibodies are likely needed to provide the best opportunity of generating a protective immune response against HIV-1. Numerous studies have demonstrated that T-cell help is necessary for effective antibody generation. However, immunogen sequences from recent HIV-1 vaccine efficacy trials include CD4 + T-cell epitopes that have evidence of immune escape. Our study shows that these epitopes, termed adapted epitopes, elicit lower frequencies of CD4 + T-cell responses in recipients from multiple HIV-1 vaccine trials. Additionally, the counterparts to these epitopes, termed nonadapted epitopes, elicited CD4 + T-cell responses that correlated with Env-specific antibodies in one efficacy trial. These results suggest that vaccine-encoded adapted epitopes dampen CD4 + T-cell responses, potentially impacting both HIV-specific antibody production and efficacious vaccine efforts.

The development of a protective vaccine remains a top priority for the control of the HIV/AIDS pandemic. Here, we show that a messenger RNA (mRNA) vaccine co-expressing membrane-anchored HIV-1 envelope (Env) and simian immunodeficiency virus (SIV) Gag proteins to generate virus-like particles (VLPs) induces antibodies capable of broad neutralization and reduces the risk of infection in rhesus macaques. In mice, immunization with co-formulated env and gag mRNAs was superior to env mRNA alone in inducing neutralizing antibodies. Macaques were primed with a transmitted-founder clade-B env mRNA lacking the N276 glycan, followed by multiple booster immunizations with glycan-repaired autologous and subsequently bivalent heterologous envs (clades A and C). This regimen was highly immunogenic and elicited neutralizing antibodies against the most prevalent (tier-2) HIV-1 strains accompanied by robust anti-Env CD4 + T cell responses. Vaccinated animals had a 79% per-exposure risk reduction upon repeated low-dose mucosal challenges with heterologous tier-2 simian-human immunodeficiency virus (SHIV AD8). Thus, the multiclade env-gag VLP mRNA platform represents a promising approach for the development of an HIV-1 vaccine., (© 2021. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)

Induction of broadly neutralizing antibodies (bNAbs) is a major goal for HIV vaccine development. HIV envelope glycoprotein (Env)-specific bNAbs isolated from HIV-infected individuals exhibit substantial somatic hypermutation and correlate with T follicular helper (Tfh) responses. Using the VC10014 DNA-protein co-immunization vaccine platform consisting of gp160 plasmids and gp140 trimeric proteins derived from an HIV-1 infected subject that developed bNAbs, we determined the characteristics of the Env-specific humoral response in vaccinated rhesus macaques in the context of CD4+ T cell depletion. Unexpectedly, both CD4+ depleted and non-depleted animals developed comparable Tier 1 and 2 heterologous HIV-1 neutralizing plasma antibody titers. There was no deficit in protection from SHIV challenge, no diminution of titers of HIV Env-specific cross-clade binding antibodies, antibody dependent cellular phagocytosis, or antibody-dependent complement deposition in the CD4+ depleted animals. These collective results suggest that in the presence of diminished CD4+ T cell help, HIV neutralizing antibodies were still generated, which may have implications for developing effective HIV vaccine strategies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Sarkar, Spencer, Barnette, Pandey, Sutton, Basu, Burch, Cleveland, Rosenberg, Rangel-Moreno, Keefer, Hessell, Haigwood and Kobie.)

Increasing evidence suggests infants develop unique neutralizing antibody (nAb) responses to HIV compared to adults. Here, we dissected the nAb response of an infant whose virus is in clinical trials as a vaccine immunogen, with a goal of characterizing the broad responses in the infant to this antigen. We isolated 73 nAbs from infant BG505 and identified a large number of clonal families. Twenty-six antibodies neutralized tier 2 viruses-in some cases, viruses from the same clade as BG505, and in others, a different clade, although none showed notable breadth. Several nAbs demonstrated antibody-dependent cellular cytotoxicity activity and targeted the V3 loop. These findings suggest an impressive polyclonal response to HIV infection in infant BG505, adding to the growing evidence that the nAb response to HIV in infants is polyclonal-a desirable vaccine response to a rapidly evolving virus like HIV., Competing Interests: The authors declare no competing interests., (© 2021 The Authors.)

A vaccine which is effective against the HIV virus is considered to be the best solution to the ongoing global HIV/AIDS epidemic. In the past thirty years, numerous attempts to develop an effective vaccine have been made with little or no success, due, in large part, to the high mutability of the virus. More recent studies showed that a vaccine able to elicit broadly neutralizing antibodies (bnAbs), that is, antibodies that can neutralize a high fraction of global virus variants, has promise to protect against HIV. Such a vaccine has been proposed to involve at least three separate stages: First, activate the appropriate precursor B cells; second, shepherd affinity maturation along pathways toward bnAbs; and, third, polish the Ab response to bind with high affinity to diverse HIV envelopes (Env). This final stage may require immunization with a mixture of Envs. In this paper, we set up a framework based on theory and modeling to design optimal panels of antigens to use in such a mixture. The designed antigens are characterized experimentally and are shown to be stable and to be recognized by known HIV antibodies., Competing Interests: The authors declare no competing interest.

The tobacco variant Nicotiana benthamiana has recently emerged as a versatile host for the manufacturing of protein therapeutics, but the fidelity of many recombinant proteins generated in this system is compromised by inadvertent proteolysis. Previous studies have revealed that the anti-HIV-1 antibodies 2F5 and PG9 as well as the protease inhibitor α 1 -antitrypsin (A1AT) are particularly susceptible to N. benthamiana proteases. Here, we identify two subtilisin-like serine proteases (NbSBT1 and NbSBT2) whose combined action is sufficient to account for all major cleavage events observed upon expression of 2F5, PG9 and A1AT in N. benthamiana. We propose that downregulation of NbSBT1 and NbSBT2 activities could constitute a powerful means to optimize the performance of this promising platform for the production of biopharmaceuticals. DATABASES: NbSBT sequence data are available in the DDBJ/EMBL/GenBank databases under the accession numbers MN534996 to MN535005., (© 2020 Federation of European Biochemical Societies.)

The membrane proximal external region (MPER) of HIV-1 gp41 is targeted by several neutralizing antibodies (NAbs) and is of interest for vaccine design. In this study, we identified novel MPER peptide mimotopes and evaluated their reactivity with HIV + plasma antibodies to characterize the diversity of the immune responses to MPER during natural infection. We utilized phage display technology to generate novel mimotopes that fit antigen-binding sites of MPER NAbs 4E10, 2F5 and Z13. Plasma antibodies from 10 HIV + patients were mapped by phage immunoprecipitation, to identify unique patient MPER binding profiles that were distinct from, and overlapping with, those of MPER NAbs. 4E10 mimotope binding profiles correlated with plasma neutralization of HIV-2/HIV-1 MPER chimeric virus, and with overall plasma neutralization breadth and potency. When administered as vaccines, 4E10 mimotopes elicited low titer NAb responses in mice. HIV mimotopes may be useful for detailed analysis of plasma antibody specificity., (Copyright © 2020. Published by Elsevier Inc.)

Non-human primates (NHP) are the only animal model suitable to evaluate the protection efficacy of HIV-1 vaccines. It is important to understand how and when neutralizing antibodies (nAbs) with specificities similar to those of human broadly neutralizing antibodies (bnAbs) develop in NHPs. To address these questions, we determined plasma neutralization specificities in two macaques which developed neutralization breadth after long-term simian/human immunodeficiency virus (SHIV) infection and identified neutralization escape mutations by analyzing the env sequences from longitudinal plasma samples. Neutralization activities targeting V2, CD4bs, V3 and gp120-gp41 interface only became detectable in week 350 plasma from macaques G1015R and G1020R using 25710 env mutants. When mapped with CAP45 env mutants, only V2 specificity was detected at week 217 and persisted until week 350 in G1015R. Neutralization escape mutations were found in CD4bs and V2 regions. However, all of them were different from those resistant mutations identified for human bnAbs. These results show that nAbs with specificities similar to human bnAbs are only detectable after long-term SHIV infection and that neutralization escape mutations in macaques are different from those found in HIV-1-infected individuals. These findings can have important implications in the best utilization of the NHP model to evaluate HIV-1 vaccines.

Soluble recombinant native-like (NL) envelope glycoprotein (Env) trimers of various human immunodeficiency virus type 1 (HIV-1) genotypes are being developed as vaccine candidates aimed at the induction of broadly neutralizing antibodies (bNAbs). The prototypic design, designated BG505 SOSIP.664, incorporates an intersubunit disulfide bond (SOS) to covalently link the gp120 and gp41 ectodomain (gp41 ECTO ) subunits and a point substitution, I559P (IP), to further stabilize the gp41 ECTO components. Without the SOS and IP changes, proteolytically cleaved trimers tend to disintegrate into their constituent gp120 and gp41 ECTO subunits. We show, however, that NL trimers lacking the SOS and/or IP change can be affinity purified in amounts sufficient for analyses of their antigenicity and thermal stability. In general, these trimer variants have properties highly comparable to those of the fully stabilized SOSIP.664 version. We conclude that the major effect of the SOS and IP changes is to substantially increase trimer stability during and after the expression process, thereby allowing useful amounts to be produced. However, once the trimers have been purified, the SOS and IP changes have only subtle impacts on thermostability and the antigenicity of bNAb and other epitopes. IMPORTANCE Recombinant trimeric proteins based on HIV-1 env genes are being developed for vaccine trials in humans. A feature of these proteins is their mimicry of the envelope glycoprotein structure on virus particles that is targeted by neutralizing antibodies, i.e., antibodies that prevent cells from becoming infected. One vaccine concept under exploration is that recombinant trimers may be able to elicit virus-neutralizing antibodies when delivered as immunogens. A commonly used design is designated SOSIP.664, a term reflecting the sequence changes that are used to stabilize the trimers and allow their production in practically useful amounts. Here, we show that these stabilizing changes act to increase trimer yield during the biosynthesis process within the producer cell but have little impact on the properties of purified trimers., (Copyright © 2019 American Society for Microbiology.)

There is an urgent need for the development of potent vaccination regimens that are able to induce specific T and B cell responses against human immunodeficiency virus type 1 (HIV-1). Here, we describe the generation and characterization of a fusion antigen comprised of the HIV-1 envelope GP120 glycoprotein from clade C (GP120C) fused at its C-terminus, with the modified vaccinia virus (VACV) 14K protein ( A27L gene) (termed GP120C14K). The design is directed toward improving the immunogenicity of the GP120C protein through its oligomerization facilitated by the fused VACV 14K protein that results in hexamer-like structures. Two different immunogens were generated: a recombinant GP120C14K fusion protein (purified from a stable CHO-K1 cell line) and a recombinant modified vaccinia virus Ankara (MVA) poxvirus vector expressing the GP120C14K fusion protein (termed MVA-GP120C14K). The GP120C14K fusion protein is recognized by broadly neutralizing antibodies (bNAbs) against HIV-1. In a murine model, a heterologous prime/boost immunization regimen with MVA-GP120C14K prime followed by adjuvanted GP120C14K protein boost generated stronger and polyfunctional HIV-1 Env-specific CD8 T cell responses when compared with the delivery of the monomeric GP120C form. Furthermore, the immunization protocol MVA-GP120C14K/GP120C14K elicited higher HIV-1 Env-specific T follicular helper cells, germinal center B cells and antibody responses than monomeric GP120. In addition, a similar MVA-GP120C14K prime/GP120C14K protein boost regimen performed in rabbits triggered high HIV-1-Env-specific IgG binding antibody titers that were capable of neutralizing HIV-1 pseudoviruses. The extent of HIV-1 neutralization was comparable to that elicited by the current standard GP140 SOSIP trimers from clades B and C when immunized as MVA-SOSIP prime/SOSIP protein boost regimen. Overall, the novel fusion antigen and the corresponding immunization scheme provided in this report can therefore be considered as potential vaccine strategies against HIV-1., (Copyright © 2019 Raman, Mejías-Pérez, Gomez, García-Arriaza, Perdiguero, Vijayan, Pérez-Ruiz, Cuervo, Santiago, Sorzano, Sánchez-Corzo, Moog, Burger, Schorcht, Sanders, Carrascosa and Esteban.)

Monoclonal antibodies (mABs) are of great biopharmaceutical importance for the diagnosis and treatment of diseases. However, their production in mammalian expression hosts usually requires extensive production times and is expensive. Escherichia coli has become a new platform for production of functional small antibody fragment variants. In this study, we have used a rhamnose-inducible expression system that allows precise control of protein expression levels. The system was first evaluated for the cytoplasmic production of super folder green fluorescence protein (sfGFP) in various production platforms and then for the periplasmic production of the anti-HIV single-chain variable antibody fragment (scFv) of PGT135. Anti-HIV broadly neutralizing antibodies, like PGT135, have potential for clinical use to prevent HIV transmission, to promote immune responses and to eradicate infected cells. Different concentrations of L-rhamnose resulted in the controlled production of both sfGFP and scFv PGT135 antibody. In addition, by optimizing the culture conditions, the amount of scFv PGT135 antibody that was expressed soluble or as inclusions bodies could be modulated. The proteins were produced in batch bioreactors, with yields of 4.9 g/L for sfGFP and 0.8 g/L for scFv. The functionality of the purified antibodies was demonstrated by their ability to neutralize a panel of different HIV variants in vitro. We expect that this expression system will prove very useful for the development of a more cost-effective production process for proteins and antibody fragments in microbial cells.

Recombinant Mycobacterium strains such as recombinant BCG (rBCG) have received considerable attention for the HIV-1 vaccine development. Recently, we described a temperature-sensitive Mycobacterium paragordonae (Mpg) strain as a novel live tuberculosis vaccine that is safer and showed an enhanced protective effect against mycobacterial infection compared to BCG. We studied the possibility of developing a vaccine against HIV-1 infection using rMpg strain expressing the p24 antigen (rMpg-p24). We observed that rMpg-p24 can induce an increased p24 expression in infected antigen presenting cells (APCs) compared to rBCG-p24. We also observed that rMpg-p24 can induce enhanced p24 specific immune responses in vaccinated mice as evidenced by increased p24-specific T lymphocyte proliferation, gamma interferon induction, antibody production and cytotoxic T lymphocyte (CTL) responses. Furthermore, an rMpg-p24 prime and plasmid DNA boost showed an increased CTL response and antibody production compared to rBCG or rMpg alone. In summary, our study indicates that a live rMpg-p24 strain induced enhanced immune responses against HIV-1 Gag in vaccinated mice. Thus, rMpg-p24 may have potential as a preventive prime vaccine in a heterologous prime-boost regimen for HIV-1 infection.

The CD4 binding site (CD4bs) of the HIV-1 envelope glycoprotein is susceptible to multiple lineages of broadly neutralizing antibodies (bnAbs) that are attractive to elicit with vaccines. The CH235 lineage (VH1-46) of CD4bs bnAbs is particularly attractive because the most mature members neutralize 90% of circulating strains, do not possess long HCDR3 regions, and do not contain insertions and deletions that may be difficult to induce. We used virus neutralization to measure the interaction of CH235 unmutated common ancestor (CH235 UCA) with functional Env trimers on infectious virions to guide immunogen design for this bnAb lineage. Two Env mutations were identified, one in loop D (N279K) and another in V5 (G458Y), that acted synergistically to render autologous CH505 transmitted/founder virus susceptible to neutralization by CH235 UCA. Man5-enriched N-glycans provided additional synergy for neutralization. CH235 UCA bound with nanomolar affinity to corresponding soluble native-like Env trimers as candidate immunogens. A cryo-EM structure of CH235 UCA bound to Man5-enriched CH505.N279K.G458Y.SOSIP.664 revealed interactions of the antibody light chain complementarity determining region 3 (CDR L3) with the engineered Env loops D and V5. These results demonstrate that virus neutralization can directly inform vaccine design and suggest a germline targeting and reverse engineering strategy to initiate and mature the CH235 bnAb lineage., Competing Interests: The authors have declared that no competing interests exist.

Self-amplifying RNA (saRNA) is a promising biotherapeutic tool that has been used as a vaccine against both infectious diseases and cancer. saRNA has been shown to induce protein expression for up to 60 days and elicit immune responses with lower dosing than messenger RNA (mRNA). Because saRNA is a large (~9500 nt), negatively charged molecule, it requires a delivery vehicle for efficient cellular uptake and degradation protection. Lipid nanoparticles (LNPs) have been widely used for RNA formulations, where the prevailing paradigm is to encapsulate RNA within the particle, including the first FDA-approved small-interfering siRNA therapy. Here, we compared LNP formulations with cationic and ionizable lipids with saRNA either on the interior or exterior of the particle. We show that LNPs formulated with cationic lipids protect saRNA from RNAse degradation, even when it is adsorbed to the surface. Furthermore, cationic LNPs deliver saRNA equivalently to particles formulated with saRNA encapsulated in an ionizable lipid particle, both in vitro and in vivo. Finally, we show that cationic and ionizable LNP formulations induce equivalent antibodies against HIV-1 Env gp140 as a model antigen. These studies establish formulating saRNA on the surface of cationic LNPs as an alternative to the paradigm of encapsulating RNA.

The discovery of broadly neutralizing antibodies (bNAbs) has been a major step towards better prophylactic and therapeutic agents against human immunodeficiency virus type 1 (HIV-1). However, effective therapy will likely require a combination of anti-HIV agents to avoid viral evasion. One possible solution to this problem is the creation of bispecific molecules that can concurrently target two vulnerable sites providing synergistic inhibitory effects. Here, we describe the production in plants and anti-HIV activity of a novel bispecific fusion protein consisting of the antigen-binding fragment (Fab) of the CD4 binding site-specific bNAb VRC01 and the antiviral lectin Avaren, which targets the glycan shield of the HIV-1 envelope (VRC01 Fab -Avaren). This combination was justified by a preliminary experiment demonstrating the synergistic HIV-1 neutralization activity of VRC01 and Fc-fused Avaren dimer (Avaren-Fc). Using the GENEWARE ® tobacco mosaic virus vector, VRC01 Fab -Avaren was expressed in Nicotiana benthamiana and purified using a three-step chromatography procedure. Surface plasmon resonance and ELISA demonstrated that both the Avaren and VRC01 Fab moieties retain their individual binding specificities. VRC01 Fab -Avaren demonstrated enhanced neutralizing activity against representative HIV-1 strains from A, B and C clades, compared to equimolar combinations of VRC01 Fab and Avaren. Notably, VRC01 Fab -Avaren showed significantly stronger neutralizing effects than the bivalent parent molecules VRC01 IgG and Avaren-Fc, with IC 50 values ranging from 48 to 310 pm. These results support the continued development of bispecific anti-HIV proteins based on Avaren and bNAbs, to which plant-based transient overexpression systems will provide an efficient protein engineering and production platform., (© 2019 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

The development of an effective vaccine against HIV infection remains a global priority. Dendritic cell (DC)-based HIV immunotherapeutic vaccine is a promising approach which aims at optimizing the HIV-specific immune response using primed DCs to promote and enhance both the cellular and humoral arms of immunity. Since the Ebola virus envelope glycoprotein (EboGP) has strong DC-targeting ability, we investigated whether EboGP is able to direct HIV particles towards DCs efficiently and promote potent HIV-specific immune responses. Our results indicate that the incorporation of EboGP into non-replicating virus-like particles (VLPs) enhances their ability to target human monocyte-derived dendritic cells (MDDCs) and monocyte-derived macrophages (MDMs). Also, a mucin-like domain deleted EboGP (EboGPΔM) can further enhanced the MDDCs and MDMs-targeting ability. Furthermore, we investigated the effect of EboGP on HIV immunogenicity in mice, and the results revealed a significantly stronger HIV-specific humoral immune response when immunized with EboGP-pseudotyped HIV VLPs compared with those immunized with HIV VLPs. Splenocytes harvested from mice immunized with EboGP-pseudotyped HIV VLPs secreted increased levels of macrophage inflammatory proteins-1α (MIP-1α) and IL-4 upon stimulation with HIV Env and/or Gag peptides compared with those harvested from mice immunized with HIV VLPs. Collectively, this study provides evidence for the first time that the incorporation of EboGP in HIV VLPs can facilitate DC and macrophage targeting and induce more potent immune responses against HIV., Competing Interests: The authors declare that they have no conflict of interest.

An array of carbohydrates masks the HIV-1 surface protein Env, contributing to the evasion of humoral immunity. In most HIV-1 isolates 'glycan holes' occur due to natural sequence variation, potentially revealing the underlying protein surface to the immune system. Here we computationally design epitopes that mimic such surface features (carbohydrate-occluded neutralization epitopes or CONE) of Env through 'epitope transplantation', in which the target region is presented on a carrier protein scaffold with preserved structural properties. Scaffolds displaying the four CONEs are examined for structure and immunogenicity. Crystal structures of two designed proteins reflect the computational models and accurately mimic the native conformations of CONEs. The sera from rabbits immunized with several CONE immunogens display Env binding activity. Our method determines essential structural elements for targets of protective antibodies. The ability to design immunogens with high mimicry to viral proteins also makes possible the exploration of new templates for vaccine development.

Limited success of current HIV-1 vaccines warrants new approaches. We discuss feasibility and potential benefits of early life HIV-1 immunization followed by vaccine boosts during childhood that may enable maturation of vaccine-induced broad anti-HIV-1 immunity over several years. By initiating this immunization approach in the very young, well before sexual debut, such a strategy may dramatically reduce the risk of HIV-1 infection.

An effective HIV-1 vaccine probably will need to be able to induce broadly neutralizing HIV-1 antibodies (bNAbs) in order to be efficacious. The many bNAbs that have been isolated from HIV-1 infected patients illustrate that the human immune system is able to elicit this type of antibodies. The elucidation of the structure of the HIV-1 envelope glycoprotein (Env) trimer has further fueled the search for Env immunogens that induce bNAbs, but while native Env trimer mimetics are often capable of inducing strain-specific neutralizing antibodies (NAbs) against the parental virus, they have not yet induced potent bNAb responses. To improve the performance of Env trimer immunogens, researchers have studied the immune responses that Env trimers have induced in animals; they have evaluated how to best use Env trimers in various immunization regimens; and they have engineered increasingly stabilized Env trimer variants. Here, we review the different approaches that have been used to increase the stability of HIV-1 Env trimer immunogens with the aim of improving the induction of NAbs. In particular, we draw parallels between the various approaches to stabilize Env trimers and ones that have been used by nature in extremophile microorganisms in order to survive in extreme environmental conditions.

Pichia pastoris (Komagataella phaffi) has been used for recombinant protein production for over 30 years with over 5000 proteins reported to date. However, yields of antibody are generally low. We have evaluated the effect of secretion signal peptides on the production of a broadly neutralizing antibody (VRC01) to increase yield. Eleven different signal peptides, including the murine IgG1 signal peptide, were combinatorially evaluated for their effect on antibody titer. Strains using different combinations of signal peptides were identified that secreted approximately 2-7 fold higher levels of VRC01 than the previous best secretor, with the highest yield of 6.50 mg L -1 in shake flask expression. Interestingly it was determined that the highest yields were achieved when the murine IgG1 signal peptide was fused to the light chain, with several different signal peptides leading to high yield when fused to the heavy chain. Finally, we have evaluated the effect of using a 2A signal peptide to create a bicistronic vector in the attempt to reduce burden and increase transformation efficiency, but found it to give reduced yields compared to using two independent vectors., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

The transmission of HIV can be prevented by the application of neutralizing monoclonal antibodies and lectins. Traditional recombinant protein manufacturing platforms lack sufficient capacity and are too expensive for developing countries, which suffer the greatest disease burden. Plants offer an inexpensive and scalable alternative manufacturing platform that can produce multiple components in a single plant, which is important because multiple components are required to avoid the rapid emergence of HIV-1 strains resistant to single microbicides. Furthermore, crude extracts can be used directly for prophylaxis to avoid the massive costs of downstream processing and purification. We investigated whether rice could simultaneously produce three functional HIV-neutralizing proteins (the monoclonal antibody 2G12, and the lectins griffithsin and cyanovirin-N). Preliminary in vitro tests showed that the cocktail of three proteins bound to gp120 and achieved HIV-1 neutralization. Remarkably, when we mixed the components with crude extracts of wild-type rice endosperm, we observed enhanced binding to gp120 in vitro and synergistic neutralization when all three components were present. Extracts of transgenic plants expressing all three proteins also showed enhanced in vitro binding to gp120 and synergistic HIV-1 neutralization. Fractionation of the rice extracts suggested that the enhanced gp120 binding was dependent on rice proteins, primarily the globulin fraction. Therefore, the production of HIV-1 microbicides in rice may not only reduce costs compared to traditional platforms but may also provide functional benefits in terms of microbicidal potency., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

A central puzzle in HIV-1 research is the inability of vaccination or even infection to reliably elicit humoral responses against broadly neutralizing epitopes in the HIV-1 envelope protein. In infected individuals, broadly neutralizing antibodies (bNAbs) do arise in a substantial minority, but only after 2 or more years of chronic infection. All known bNAbs possess at least one of three traits: a high frequency of somatic hypermutation, a long third complementarity determining region in the antibody heavy chain (HCDR3), or significant poly- or autoreactivity. Collectively, these observations suggest a plausible explanation for the rarity of many types of bNAbs: namely, that their generation is blocked by immunological tolerance or immune response checkpoints, thereby mandating that B cells take a tortuous path of somatic evolution over several years to achieve broadly neutralizing activity. In this brief review, we discuss the evidence for this tolerance hypothesis, its implications for HIV-1 vaccine design, and potential ways to access normally forbidden compartments of the antibody repertoire by modulating or circumventing tolerance controls.

The elicitation of broadly neutralizing antibodies (bnAbs) is considered crucial for an effective, preventive HIV-1 vaccine. Led by the discovery of a new generation of potent bnAbs, the field has significantly advanced over the past decade. There is a wealth of knowledge about the development of bnAbs in natural infection, their specificity, potency, breadth and function. Yet, devising immunogens and vaccination regimens that evoke bnAb responses has not been successful. Where are the roadblocks in their development? What can we learn from natural infection, where bnAb induction is possible but rare? Herein, we will reflect on key discoveries and discuss open questions that may bear crucial insights needed to move towards creating effective bnAb vaccines.

Developing an effective prophylaxis HIV-1 vaccine is likely to require the elicitation of broadly neutralizing antibodies (bnAbs). As the HIV-1 envelope (Env) glycoprotein - the sole target of bnAbs - has evolved multiple mechanisms to evade antibody neutralization, the processes for bnAb generation are highly selective and time-consuming. Benefiting from antibody isolation technologies of single B cell culturing and direct single B cell sorting and cloning, a new generation of monoclonal bnAbs has been isolated since 2009, exhibiting remarkable breadths and potencies, thus breaking through a nearly 20-year-long limit of four monoclonal bnAbs with moderate breadth and potency. The discovery of a long list of monoclonal bnAbs has provided in-depth understanding of the sites of vulnerability on the HIV-1 Env and the complexity of human B cell immunology to generate such responses, thus presenting both guidance and challenges to move the Env immunogen design effort forward.

Some of the strongest immune correlates of controlled HIV infection include markers related to antiviral T-cell responses, especially responses mediated by CD8+ cytotoxic T lymphocytes (CTL). These observations and lessons learned from other viral infections have motivated the development of T-cell vaccine candidates to HIV in the preventive and especially in the therapeutic setting. While none of the T-cell vaccine concepts tested to date have shown sufficient efficacy, the last few years have seen some advances indicating that strategies activating the cellular adaptive immune response to HIV may present a critical component of an effective therapeutic and possibly preventive HIV vaccine. Some of the important components that a successful T-cell vaccine may need to contain and additional considerations for the design and delivery of such vaccines are discussed in this chapter.

Despite progress in antiretroviral therapy, pre-exposure prophylaxis, microbicides, and other preventive strategies, a vaccine to prevent HIV-1 infection remains desperately needed. Development of an effective vaccine is challenged by several immunologic features of HIV-1 evidenced by the failure of five of the six HIV-1 candidate vaccine efficacy trials to date. This chapter reviews these efficacy trials with a focus on the Phase 3 RV144 trial in Thailand, the only HIV-1 vaccine efficacy trial to show a moderate protective effect of 31% with respect to placebo administration. Although modest, this protection has allowed for the study of potential immunologic correlates of protection to improve development of future HIV-1 pox-protein and other vaccine strategies. Trials in Thailand and South Africa have built upon the RV144 framework to provide additional immunologic insights which enable current and future efficacy testing of related vaccine candidates.

A strategy for HIV-1 vaccine development is to define envelope (Env) evolution of broadly neutralizing antibodies (bnAbs) in infection and to recreate those events by vaccination. Here, we report host tolerance mechanisms that limit the development of CD4-binding site (CD4bs), HCDR3-binder bnAbs via sequential HIV-1 Env vaccination. Vaccine-induced macaque CD4bs antibodies neutralize 7% of HIV-1 strains, recognize open Env trimers, and accumulate relatively modest somatic mutations. In naive CD4bs, unmutated common ancestor knock-in mice Env + B cell clones develop anergy and partial deletion at the transitional to mature B cell stage, but become Env - upon receptor editing. In comparison with repetitive Env immunizations, sequential Env administration rescue anergic Env + (non-edited) precursor B cells. Thus, stepwise immunization initiates CD4bs-bnAb responses, but immune tolerance mechanisms restrict their development, suggesting that sequential immunogen-based vaccine regimens will likely need to incorporate strategies to expand bnAb precursor pools.

While broadly neutralizing antibodies (bnAbs) are a promising preventative and therapeutic tool for HIV infection, production is difficult and expensive. Production of antibody-like fragments in bacterial cytoplasm provides a cheaper alternative. This work explored the transplantation of the complementarity determining regions of the anti-HIV bnAbs PGT121 and 10E8 onto a single-chain variable fragment (scFv) scaffold, previously discovered through a novel screening platform. The scaffolded 10E8 scFv, but not the scaffolded PGT121 scFv, was soluble in bacterial cytoplasm, enabling efficient production in bacteria. Three additional multimeric constructs employing the scaffolded 10E8 scFv were also generated and soluble versions produced in bacteria. However, the constructs were found to have substantially lost anti-HIV binding function and had completely abrogated neutralizing activity. Overall, while this study provides a proof-of-concept for anti-HIV bnAb construct production in bacterial cytoplasm, future refinement of these technologies will be required to realize the goal of producing inexpensive and effective bnAb-like tools for the control of HIV.

A study of the induction of broadly neutralizing antibodies (bNAbs) in HIV-infected patients and vaccinated subjects revealed the main criteria for the formation of bNAbs (the duration of exposure to a viral antigen, the effect of the diversity of HIV variants, and the removal of barriers associated with the Env-dependent defense mechanisms of HIV-1). Modified trimers of the HIV-1 envelope protein (Env) exposed on virus-like particles (VLP) have unique properties: they (i) modulate the exposure of binding sites (bs) of the CD4 receptor and co-receptor; (ii) create steric restrictions for contact with bNAbs; (iii) increase the Env presentation density, thus enhancing the immune response; (iv) form stable trimers that do not induce off-target immune responses; and (v) allow additional modifications to their structure and construction of a platform with immunostimulating molecules. Immunization using a heterologous subtype-cross prime-boost regime with modified trimeric Env is capable of inducing somatic hypermutation levels necessary for the formation of bNAbs.

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., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

This review discusses recent progress made in developing a vaccine and novel treatments for human immunodeficiency virus (HIV). It highlights the shortcomings of the RV144 vaccination trial [ALVAC-HIV (vCP1521) and AIDSVAX B/E] and the current standard of care and proposes that engineered expression of broadly neutralizing antibodies (bNAbs) against HIV-1 could overcome these shortcomings. Current developments in three major lines of research on HIV prevention and treatment using bNAbs are reviewed: firstly, the use of sequential immunogens to activate B cells to express bNAbs; secondly, the delivery of novel and extremely potent bNAbs through passive administration; and finally, the use of gene transfer using adeno-associated viral vectors to deliver bNAbs.

The extreme HIV diversity posts a great challenge on development of an effective anti-HIV vaccine. To solve this problem, it is crucial to discover an appropriate immunogens and strategies that are able to prevent the transmission of the diverse viruses that are circulating in the world. Even though there have been a number of broadly neutralizing anti-HIV antibodies (bNAbs) been discovered in recent years, induction of such antibodies to date has only been observed in HIV-1 infection. Here, in this mini review, we review the progress in development of HIV vaccine in eliciting broad immune response, especially production of bNAbs, discuss possible strategies, such as polyvalent sequential vaccination, that facilitates B cell maturation leading to bNAb response.

Vesicular stomatitis virus (VSV), like many other Rhabdoviruses, have become the focus of intense research over the past couple of decades based on their suitability as vaccine vectors, transient gene delivery systems, and as oncolytic viruses for cancer therapy. VSV as a vaccine vector platform has multiple advantages over more traditional viral vectors including low level, non-pathogenic replication in diverse cell types, ability to induce both humoral and cell-mediate immune responses, and the remarkable expression of foreign proteins cloned into multiple intergenic sites in the VSV genome. The utility and safety of VSV as a vaccine vector was recently demonstrated near the end of the recent Ebola outbreak in West Africa where VSV pseudotyped with the Ebola virus (EBOV) glycoprotein was proven safe in humans and provided protective efficacy against EBOV in a human phase III clinical trial. A team of Canadian scientists, led by Dr. Gary Kobinger, is now working with International AIDS Vaccine Initiative (IAVI) in developing a VSV-based HIV vaccine that will combine unique Canadian research on the HIV-1 Env glycoprotein and on the VSV vaccine vector. The goal of this collaboration is to develop a vaccine with a robust and potent anti-HIV immune response with an emphasis on generating quality antibodies to protect against HIV challenges.

Despite the widespread use of antiretrovirals (ARV), more than 150,000 pediatric HIV-1 infections continue to occur annually. Supplemental strategies are necessary to eliminate pediatric HIV infections. We previously reported that maternal HIV envelope-specific anti-V3 IgG and CD4 binding site-directed antibodies, as well as tier 1 virus neutralization, predicted a reduced risk of mother-to-child transmission (MTCT) of HIV-1 in the pre-ARV era U.S.-based Women and Infants Transmission Study (WITS) cohort. As the majority of ongoing pediatric HIV infections occur in sub-Saharan Africa, we sought to determine if the same maternal humoral immune correlates predicted MTCT in a subset of the Malawian Breastfeeding, Antiretrovirals, and Nutrition (BAN) cohort of HIV-infected mothers ( n = 88, with 45 transmitting and 43 nontransmitting). Women and infants received ARV at delivery; thus, the majority of MTCT was in utero (91%). In a multivariable logistic regression model, neither maternal anti-V3 IgG nor clade C tier 1 virus neutralization was associated with MTCT. Unexpectedly, maternal CD4 binding-site antibodies and anti-variable loop 1 and 2 (V1V2) IgG were associated with increased MTCT, independent of maternal viral load. Neither infant envelope (Env)-specific IgG levels nor maternal IgG transplacental transfer efficiency was associated with transmission. Distinct humoral immune correlates of MTCT in the BAN and WITS cohorts could be due to differences between transmission modes, virus clades, or maternal antiretroviral use. The association between specific maternal antibody responses and in utero transmission, which is distinct from potentially protective maternal antibodies in the WITS cohort, underlines the importance of investigating additional cohorts with well-defined transmission modes to understand the role of antibodies during HIV-1 MTCT., (Copyright © 2017 American Society for Microbiology.)

Preventing the spread of infectious diseases remains an urgent priority worldwide, and this is driving the development of advanced nanotechnology to diagnose infections at the point of care. Herein, we report the creation of a library of novel nanobody capture ligands to detect p24, one of the earliest markers of HIV infection. We demonstrate that these nanobodies, one tenth the size of conventional antibodies, exhibit high sensitivity and broad specificity to global HIV-1 subtypes. Biophysical characterization indicates strong 690 pM binding constants and fast kinetic on-rates, 1 to 2 orders of magnitude better than monoclonal antibody comparators. A crystal structure of the lead nanobody and p24 was obtained and used alongside molecular dynamics simulations to elucidate the molecular basis of these enhanced performance characteristics. They indicate that binding occurs at C-terminal helices 10 and 11 of p24, a negatively charged region of p24 complemented by the positive surface of the nanobody binding interface involving CDR1, CDR2, and CDR3 loops. Our findings have broad implications on the design of novel antibodies and a wide range of advanced biomedical applications.

Mechanisms responsible for natural control of human immunodeficiency type 1 (HIV) replication in elite controllers (EC) remain incompletely defined. To determine if EC generate high quality HIV-specific IgA responses, we used Western blotting to compare the specificities and frequencies of IgA to HIV antigens in serum of gender-, age- and race-matched EC and aviremic controllers (HC) and viremic noncontrollers (HN) on highly active antiretroviral therapy (HAART). Concentrations and avidity of IgA to HIV antigens were measured using ELISA or multiplex assays. Measurements for IgG were performed in parallel. EC were found to have stronger p24- and V1V2-specific IgG responses than HN, but there were no IgG differences for EC and HC. In contrast, IgA in EC serum bound more frequently to gp160 and gag proteins than IgA in HC or HN. The avidity of anti-gp41 IgA was also greater in EC, and these subjects had stronger IgA responses to the gp41 heptad repeat region 1 (HR1), a reported target of anti-bacterial RNA polymerase antibodies that cross react with gp41. However, EC did not demonstrate greater IgA responses to E. coli RNA polymerase or to peptides containing the shared LRAI sequence, suggesting that most of their HR1-specific IgA antibodies were not induced by intestinal microbiota. In both EC and HAART recipients, the concentrations of HIV-specific IgG were greater than HIV-specific IgA, but their avidities were comparable, implying that they could compete for antigen. Exceptions were C1 peptides and V1V2 loops. IgG and IgA responses to these antigens were discordant, with IgG reacting to V1V2, and IgA reacting to C1, especially in EC. Interestingly, EC with IgG hypergammaglobulinemia had greater HIV-specific IgA and IgG responses than EC with normal total IgG levels. Heterogeneity in EC antibody responses may therefore be due to a more focused HIV-specific B cell response in some of these individuals. Overall, these data suggest that development of HIV-specific IgA responses and affinity maturation of anti-gp41 IgA antibodies occurs to a greater extent in EC than in subjects on HAART. Future studies will be required to determine if IgA antibodies in EC may contribute in control of viral replication.

Soluble, recombinant native-like envelope glycoprotein (Env) trimers of various human immunodeficiency virus type 1 (HIV-1) genotypes are being developed for structural studies and as vaccine candidates aimed at the induction of broadly neutralizing antibodies (bNAbs). The prototypic design is designated SOSIP.664, but many HIV-1 env genes do not yield fully native-like trimers efficiently. One such env gene is CZA97.012 from a neutralization-resistant (tier 2) clade C virus. As appropriately purified, native-like CZA97.012 SOSIP.664 trimers induce autologous neutralizing antibodies (NAbs) efficiently in immunized rabbits, we sought to improve the efficiency with which they can be produced and to better understand the limitations to the original design. By using structure- and antigenicity-guided mutagenesis strategies focused on the V2 and V3 regions and the gp120-gp41 interface, we developed the CZA97 SOSIP.v4.2-M6.IT construct. Fully native-like, stable trimers that display multiple bNAb epitopes could be expressed from this construct in a stable CHO cell line and purified at an acceptable yield using either a PGT145 or a 2G12 bNAb affinity column. We also show that similar mutagenesis strategies can be used to improve the yields and properties of SOSIP.664 trimers of the DU422, 426c, and 92UG037 genotypes. IMPORTANCE Recombinant trimeric proteins based on HIV-1 env genes are being developed for future vaccine trials in humans. A feature of these proteins is their mimicry of the envelope glycoprotein (Env) structure on virus particles that is targeted by neutralizing antibodies, i.e., antibodies that prevent cells from becoming infected. The vaccine concept under exploration is that recombinant trimers may be able to elicit virus-neutralizing antibodies when delivered as immunogens. Because HIV-1 is extremely variable, a practical vaccine may need to incorporate Env trimers derived from multiple different virus sequences. Accordingly, we need to understand how to make recombinant trimers from many different env genes. Here, we show how to produce trimers from a clade C virus, CZA97.012, by using an array of protein engineering techniques to improve a prototypic construct. We also show that the methods may have wider utility for other env genes, thereby further guiding immunogen design., (Copyright © 2017 American Society for Microbiology.)

An HIV-1 vaccine is needed to curtail the HIV epidemic. Only one (RV144) out of the 6 HIV-1 vaccine efficacy trials performed showed efficacy. A potential mechanism of protection is the induction of functional antibodies to V1V2 region of HIV envelope. The 2 main current approaches to the generation of protective immunity are through broadly neutralizing antibodies (bnAb) and induction of functional antibodies (non-neutralizing Abs with other potential anti-viral functions). Passive immunization using bnAb has advanced into phase II clinical trials. The induction of bnAb using mimics of the natural Env trimer or B-cell lineage vaccine design is still in pre-clinical phase. An attempt at optimization of protective functional antibodies will be assessed next with the efficacy trial (HVTN702) about to start. With on-going optimization of prime/boost strategies, the development of mosaic immunogens, replication competent vectors, and emergence of new strategies designed to induce bnAb, the prospects for a preventive HIV vaccine have never been more promising.

Previously, VLPs bearing JR-FL strain HIV-1 Envelope trimers elicited potent neutralizing antibodies (nAbs) in 2/8 rabbits (PLoS Pathog 11(5): e1004932) by taking advantage of a naturally absent glycan at position 197 that borders the CD4 binding site (CD4bs). In new immunizations, we attempted to improve nAb responses by removing the N362 glycan that also lines the CD4bs. All 4 rabbits developed nAbs. One targeted the N197 glycan hole like our previous sera. Two sera depended on the N463 glycan, again suggesting CD4bs overlap. Heterologous boosts appeared to reduce nAb clashes with the N362 glycan. The fourth serum targeted a N362 glycan-sensitive epitope. VLP manufacture challenges prevented us from immunizing larger rabbit numbers to empower a robust statistical analysis. Nevertheless, trends suggest that targeted glycan removal may improve nAb induction by exposing new epitopes and that it may be possible to modify nAb specificity using rational heterologous boosts., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Monoclonal antibodies are one of the fastest growing classes of pharmaceutical products, however, their potential is limited by the high cost of development and manufacturing. Here we present a safe and cost-effective platform for in vivo expression of therapeutic antibodies using nucleoside-modified mRNA. To demonstrate feasibility and protective efficacy, nucleoside-modified mRNAs encoding the light and heavy chains of the broadly neutralizing anti-HIV-1 antibody VRC01 are generated and encapsulated into lipid nanoparticles. Systemic administration of 1.4 mg kg -1 of mRNA into mice results in ∼170 μg ml -1 VRC01 antibody concentrations in the plasma 24 h post injection. Weekly injections of 1 mg kg -1 of mRNA into immunodeficient mice maintain trough VRC01 levels above 40 μg ml -1 . Most importantly, the translated antibody from a single injection of VRC01 mRNA protects humanized mice from intravenous HIV-1 challenge, demonstrating that nucleoside-modified mRNA represents a viable delivery platform for passive immunotherapy against HIV-1 with expansion to a variety of diseases.

Confirmatory tests using Western blot (WB) and HIV-1 nucleic acid testing (HIV-1 RNA) following a positive screening test are required for the diagnosis of HIV-1 infection according to the current Japanese guidelines for HIV-1/2 diagnosis. We report herein on a rare case in a patient who remained negative for WB over 10 months in spite of being positive by fourth-generation immunoassays (4thGIA) and who subsequently seroreverted by 4thGIA for three months after initiating antiretroviral therapy., Case: A man in his early twenties previously visited a hospital because of fever in October 2012. Laboratory data revealed leukocytopenia, thrombocytopenia and increased serum ferritin, suggesting hemophagocytic syndrome (HPS). During that visit, he tested positive for a 4thGIA, but negative for HIV-1 WB and his result of HIV-1 RNA result was detected invalid because of the presence of some inhibitory material in his RNA preparation. Thereafter, he was diagnosed as having cytomegalovirus-associated HPS treatment was for which initiated. In January 2013, he developed Pneumocystis jirovecii pneumonia, and his HIV-1 RNA viral load was 7.7 × 105 copies/mL in February 2013. Acute HIV infection was suspected, because the HIV-1 WB remained negative. He was started on antiretroviral therapy in April 2013. His 4thGIA was converted to negative in May 2013 and was reconverted to positive in August 2013. HIV-1 WB, however, continued to be indeterminant until February 2014, in which it turned positive for the first time according to the CDC criteria., Methods and Results: The genetic analyses of HIV-1 were done on the gag, env, nef and pol region of the HIV-1 gene from the patient. There was no clear element to delay antibody production on the virus side. Preserved specimens of the patient were measured with eight kinds of HIV screening assay. It was thought that the fourth generation assay was positive only by the presence of the antigen until March 2013 because the antibody had not been detected., Discussion: We encountered a case of acute HIV infection in which the WB result was negative for 10 months after the first positive response of the 4thGIA. The 4thGIA is essential for the early diagnosis and early treatment of HIV infection; therefore, the 4thGIA should be strictly recommended to avoid the use of older generations of immunoassay in the diagnostic guidelines. The role of the WB test should be examined closely from various aspects for use as a confirmatory test under recent laboratory situations in which highly sensitive and specific methods, e.g. the 4th GIA, have become available. In addition, unnecessary confusion due to the diversities of antibody formation should be avoided. The antibody detection tests for HIV are still necessary and indispensable for the confirmation of the disease or the diagnosis of the acute infection stage. Therefore development of a newer antibody measuring method which could achieve an easier operation and should have a higher sensitivity and specificity for HIV confirmation is strongly expected.

Definition of the key parameters mediating effective antibody blocking of HIV-1 acquisition within mucosal tissue may prove critical to effective vaccine development and the prophylactic use of monoclonal antibodies. Although direct antibody-mediated neutralization is highly effective against cell-free virus, antibodies targeting different sites of envelope vulnerability may display differential activity against mucosal infection. Nonneutralizing antibodies (nnAbs) may also impact mucosal transmission events through Fc-gamma receptor (FcγR)-mediated inhibition. In this study, a panel of broadly neutralizing antibodies (bnAbs) and nnAbs, including those associated with protection in the RV144 vaccine trial, were screened for the ability to block HIV-1 acquisition and replication across a range of cellular and mucosal tissue models. Neutralization potency, as determined by the TZM-bl infection assay, did not fully predict activity in mucosal tissue. CD4-binding site (CD4bs)-specific bnAbs, in particular VRC01, were consistent in blocking HIV-1 infection across all cellular and tissue models. Membrane-proximal external region (MPER) (2F5) and outer domain glycan (2G12) bnAbs were also efficient in preventing infection of mucosal tissues, while the protective efficacy of bnAbs targeting V1-V2 glycans (PG9 and PG16) was more variable. In contrast, nnAbs alone and in combinations, while active in a range of cellular assays, were poorly protective against HIV-1 infection of mucosal tissues. These data suggest that tissue resident effector cell numbers and low FcγR expression may limit the potential of nnAbs to prevent establishment of the initial foci of infection. The solid protection provided by specific bnAbs clearly demonstrates their superior potential over that of nonneutralizing antibodies for preventing HIV-1 infection at the mucosal portals of infection., Importance: Key parameters mediating effective antibody blocking of HIV-1 acquisition within mucosal tissue have not been defined. While bnAbs are highly effective against cell-free virus, they are not induced by current vaccine candidates. However, nnAbs, readily induced by vaccines, can trigger antibody-dependent cellular effector functions, through engagement of their Fc-gamma receptors. Fc-mediated antiviral activity has been implicated as a secondary correlate of decreased HIV-1 risk in the RV144 vaccine efficacy trial, suggesting that protection might be mediated in the absence of classical neutralization. To aid vaccine design and selection of antibodies for use in passive protection strategies, we assessed a range of bnAbs and nnAbs for their potential to block ex vivo challenge of mucosal tissues. Our data clearly indicate the superior efficacy of neutralizing antibodies in preventing mucosal acquisition of infection. These results underscore the importance of maintaining the central focus of HIV-1 vaccine research on the induction of potently neutralizing antibodies., (Copyright © 2016 Cheeseman et al.)

The V1V2 region of HIV-1 gp120 harbors a major vulnerable site targeted by a group of broadly neutralizing monoclonal antibodies (MAbs) such as PG9 through strand-strand recognition. However, this epitope region is structurally polymorphic as it can also form a helical conformation recognized by RV144 vaccine-induced MAb CH58. This structural polymorphism is a potential mechanism for masking the V1V2 vulnerable site. Designing immunogens that can induce conformation-specific antibody (Ab) responses may lead to vaccines targeting this vulnerable site. We designed a panel of immunogens engrafting the V1V2 domain into trimeric and pentameric scaffolds in structurally constrained conformations. We also fused V1V2 to an Fc fragment to mimic the unconstrained V1V2 conformation. We tested these V1V2-scaffold proteins for immunogenicity in rabbits and assessed the responses by enzyme-linked immunosorbent assay (ELISA) and competition assays. Our V1V2 immunogens induced distinct conformation-specific Ab responses. Abs induced by structurally unconstrained immunogens reacted preferentially with unconstrained V1V2 antigens, suggesting recognition of the helical configuration, while Abs induced by the structurally constrained immunogens reacted preferentially with constrained V1V2 antigens, suggesting recognition of the β-strand conformation. The Ab responses induced by the structurally constrained immunogens were more broadly reactive and had higher titers than those induced by the structurally unconstrained immunogens. Our results demonstrate that immunogens presenting the different structural conformations of the gp120 V1V2 vulnerable site can be designed and that these immunogens induce distinct Ab responses with epitope conformation specificity. Therefore, these structurally constrained V1V2 immunogens are vaccine prototypes targeting the V1V2 domain of the HIV-1 envelope., Importance: The correlates analysis of the RV144 HIV-1 vaccine trial suggested that the presence of antibodies to the V1V2 region of HIV-1 gp120 was responsible for the modest protection observed in the trial. In addition, V1V2 harbors one of the key vulnerable sites of HIV-1 Env recognized by a family of broadly neutralizing MAbs such as PG9. Thus, V1V2 is a key target for vaccine development. However, this vulnerable site is structurally polymorphic, and designing immunogens that present different conformations is crucial for targeting this site. We show here that such immunogens can be designed and that they induced conformation-specific antibody responses in rabbits. Our immunogens are therefore prototypes of vaccine candidates targeting the V1V2 region of HIV-1 Env., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Strong antibody (Ab) responses against V1V2 epitopes of the human immunodeficiency virus type 1 (HIV-1) gp120 envelope (Env) correlated with reduced infection rates in studies of HIV, simian-human immunodeficiency virus (SHIV), and simian immunodeficiency virus (SIV). In order to focus the Ab response on V1V2, we used six V1V2 sequences and nine scaffold proteins to construct immunogens which were tested using various immunization regimens for their ability to induce cross-reactive and biologically active V2 Abs in rabbits. A prime/boost immunization strategy was employed using gp120 DNA and various V1V2-scaffold proteins. The rabbit polyclonal Ab responses (i) were successfully focused on the V1V2 region, with weak or only transient responses to other Env epitopes, (ii) displayed broad cross-reactive binding activity with gp120s and the V1V2 regions of diverse strains from clades B, C, and E, (iii) included V2 Abs with specificities similar to those found in HIV-infected individuals, and (iv) remained detectable ≥1 year after the last boosting dose. Importantly, sera from rabbits receiving V1V2-scaffold immunogens displayed Ab-dependent cellular phagocytosis whereas sera from rabbits receiving only gp120 did not. The results represent the first fully successful example of reverse vaccinology in the HIV vaccine field with rationally designed epitope scaffold immunogens inducing Abs that recapitulate the epitope specificity and biologic activity of the human monoclonal Abs from which the immunogens were designed. Moreover, this is the first immunogenicity study using epitope-targeting, rationally designed vaccine constructs that induced an Fc-mediated activity associated with protection from infection with HIV, SIV, and SHIV., Importance: Novel immunogens were designed to focus the antibody response of rabbits on the V1V2 epitopes of HIV-1 gp120 since such antibodies were associated with reduced infection rates of HIV, SIV, and SHIV. The vaccine-induced antibodies were broadly cross-reactive with the V1V2 regions of HIV subtypes B, C and E and, importantly, facilitated Fc-mediated phagocytosis, an activity not induced upon immunization of rabbits with gp120. This is the first immunogenicity study of vaccine constructs that focuses the antibody response on V1V2 and induces V2-specific antibodies with the ability to mediate phagocytosis, an activity that has been associated with protection from infection with HIV, SIV, and SHIV., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

The HIV envelope glycoprotein (Env) is extensively modified with host-derived N-linked glycans. The high density of glycosylation on the viral spike limits enzymatic processing, resulting in numerous underprocessed oligomannose-type glycans. This extensive glycosylation not only shields conserved regions of the protein from the immune system but also acts as a target for anti-HIV broadly neutralizing antibodies (bnAbs). In response to the host immune system, the HIV glycan shield is constantly evolving through mutations affecting both the positions and numbers of potential N-linked glycosylation sites (PNGSs). Here, using longitudinal Env sequences from a clade C-infected individual (CAP256), we measured the impact of the shifting glycan shield during HIV infection on the abundance of oligomannose-type glycans. By analyzing the intrinsic mannose patch from a panel of recombinant CAP256 gp120s displaying high protein sequence variability and changes in PNGS number and positioning, we show that the intrinsic mannose patch persists throughout the course of HIV infection and correlates with the number of PNGSs. This effect of the glycan density on the processing state was also supported by the analysis of a cross-clade panel of recombinant gp120 glycoproteins. Together, these observations underscore the importance of glycan clustering for the generation of carbohydrate epitopes for anti-HIV bnAbs. The persistence of the intrinsic mannose patch over the course of HIV infection further highlights this epitope as an important target for HIV vaccine strategies., Importance: Development of an HIV vaccine is critical for control of the HIV pandemic, and elicitation of broadly neutralizing antibodies (bnAbs) is likely to be a key component of a successful vaccine response. The HIV envelope glycoprotein (Env) is covered in an array of host-derived N-linked glycans often referred to as the glycan shield. This glycan shield is a target for many of the recently isolated anti-HIV bnAbs and is therefore under constant pressure from the host immune system, leading to changes in both glycan site frequency and location. This study aimed to determine whether these genetic changes impacted the eventual processing of glycans on the HIV Env and the susceptibility of the virus to neutralization. We show that despite this variation in glycan site positioning and frequency over the course of HIV infection, the mannose patch is a conserved feature throughout, making it a stable target for HIV vaccine design., (Copyright © 2016 Coss et al.)

Background: A major challenge to HIV eradication strategies is the lack of an accurate measurement of the total burden of replication-competent HIV (the "reservoir"). We assessed the association of anti-HIV antibody responses and the estimated size of the reservoir during antiretroviral therapy (ART)., Methods: We evaluated anti-HIV antibody profiles using luciferase immunoprecipitation systems (LIPS) assay in relation to several blood-based HIV reservoir measures: total and 2-LTR DNA (rtPCR or droplet digital PCR); integrated DNA (Alu PCR); unspliced RNA (rtPCR), multiply-spliced RNA (TILDA), residual plasma HIV RNA (single copy PCR), and replication-competent virus (outgrowth assay). We also assessed total HIV DNA and RNA in gut-associated lymphoid tissue (rtPCR). Spearman correlations and linear regressions were performed using log-transformed blood- or tissue-based reservoir measurements as predictors and log-transformed antibody levels as outcome variables., Results: Among 51 chronically HIV-infected ART-suppressed participants (median age = 57, nadir CD4+ count = 196 cells/mm3, ART duration = 9 years), the most statistically significant associations were between antibody responses to integrase and HIV RNA in gut-associated lymphoid tissue (1.17 fold-increase per two-fold RNA increase, P = 0.004) and between antibody responses to matrix and integrated HIV DNA in resting CD4+ T cells (0.35 fold-decrease per two-fold DNA increase, P = 0.003). However, these associations were not statistically significant after a stringent Bonferroni-adjustment of P<0.00045. Multivariate models including age and duration of ART did not markedly alter results., Conclusions: Our findings suggest that anti-HIV antibody responses may reflect the size of the HIV reservoir during chronic treated HIV disease, possibly via antigen recognition in reservoir sites. Larger, prospective studies are needed to validate the utility of antibody levels as a measure of the total body burden of HIV during treatment.

After three decades of intensive research efforts, an effective vaccine against HIV-1 remains to be developed. Several broadly neutralizing antibodies to HIV-1, such as 10E8, recognize the membrane proximal external region (MPER) of the HIV-1 gp41 protein. Thus, the MPER is considered to be a very important target for vaccine design. However, the MPER segment has very weak immunogenicity and tends to insert its epitope residues into the cell membrane, thereby avoiding antibody binding. To address this complication in vaccine development, we herein designed a peptide, designated 10E8-4P, containing four copies of the 10E8 epitope as an immunogen. As predicted by structural simulation, 10E8-4P exhibits a well-arranged tandem helical conformation, with the key residues in the 10E8 epitope oriented at different angles, thus suggesting that some of these key residues may be exposed outside of the lipid membrane. Compared with a peptide containing a single 10E8 epitope (10E8-1P), 10E8-4P not only exhibited better antigenicity but also elicited neutralizing antibody response against HIV-1 pseudoviruses, whereas 10E8-1P could not induce detectable neutralizing antibody response. Importantly, antibodies elicited by 10E8-4P also possessed a strong ability to activate an antibody-dependent cell-mediated cytotoxicity (ADCC) reporter gene, thus suggesting that they may have ADCC activity. Therefore, this strategy shows promise for further optimization and application in future HIV-1 vaccine design.

The majority of new Human Immunodeficiency Virus (HIV)-1 infections are acquired via sexual transmission at mucosal surfaces. Partial efficacy (31.2%) of the Thai RV144 HIV-1 vaccine trial has been correlated with Antibody-dependent Cellular Cytotoxicity (ADCC) mediated by non-neutralizing antibodies targeting the V1V2 region of the HIV-1 envelope. This has led to speculation that ADCC and other antibody-dependent cellular effector functions might provide an important defense against mucosal acquisition of HIV-1 infection. However, the ability of antibody-dependent cellular effector mechanisms to impact on early mucosal transmission events will depend on a variety of parameters including effector cell type, frequency, the class of Fc-Receptor (FcR) expressed, the number of FcR per cell and the glycoslyation pattern of the induced antibodies. In this study, we characterize and compare the frequency and phenotype of IgG (CD16 [FcγRIII], CD32 [FcγRII] and CD64 [FcγRI]) and IgA (CD89 [FcαR]) receptor expression on effector cells within male and female genital mucosal tissue, colorectal tissue and red blood cell-lysed whole blood. The frequency of FcR expression on CD14+ monocytic cells, myeloid dendritic cells and natural killer cells were similar across the three mucosal tissue compartments, but significantly lower when compared to the FcR expression profile of effector cells isolated from whole blood, with many cells negative for all FcRs. Of the three tissues tested, penile tissue had the highest percentage of FcR positive effector cells. Immunofluorescent staining was used to determine the location of CD14+, CD11c+ and CD56+ cells within the three mucosal tissues. We show that the majority of effector cells across the different mucosal locations reside within the subepithelial lamina propria. The potential implication of the observed FcR expression patterns on the effectiveness of FcR-dependent cellular effector functions to impact on the initial events in mucosal transmission and dissemination warrants further mechanistic studies.

Follicular helper T (Tfh) cells provide crucial help to germinal center B (GCB) cells for proper antibody production, and a specialized subset of regulatory T cells, follicular regulatory T (Tfr) cells, modulate this process. However, Tfr-cell function in the GC is not well understood. Here, we define Tfr cells as a CD4(+) Foxp3(+) CXCR5(hi) PD-1(hi) CD25(low) TIGIT(high) T-cell population. Furthermore, we have used a novel mouse model ("Bcl6FC") to delete the Bcl6 gene in Foxp3(+) T cells and thus specifically deplete Tfr cells. Following immunization, Bcl6FC mice develop normal Tfh- and GCB-cell populations. However, Bcl6FC mice produce altered antigen-specific antibody responses, with reduced titers of IgG and significantly increased IgA. Bcl6FC mice also developed IgG antibodies with significantly decreased avidity to antigen in an HIV-1 gp120 "prime-boost" vaccine model. In an autoimmune lupus model, we observed strongly elevated anti-DNA IgA titers in Bcl6FC mice. Additionally, Tfh cells from Bcl6FC mice consistently produce higher levels of Interferon-γ, IL-10 and IL-21. Loss of Tfr cells therefore leads to highly abnormal Tfh-cell and GCB-cell responses. Overall, our study has uncovered unique regulatory roles for Tfr cells in the GC response., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

An ideal protective HIV-1 vaccine can elicit broadly neutralizing antibodies, capable of preventing HIV transmission. The strategies of designing vaccines include generation of soluble recombinant proteins which mimic the native Env complex and are able to enhance the immunogenicity of gp120. Recent data indicate that the cytoplasmic tail (CT) of the Env protein has multiple functions, which can affect the early steps of infection, as well as viral assembly and antigenic properties. Modifications in the CT can be used to induce conformational changes in functional regions of gp120 and to stabilize the trimeric structure, avoiding immune misdirection and induction of non-neutralizing antibody responses. Env-trimers with modified CTs in virus-like particles (VLPs) are able to induce antibodies with broad spectrum neutralizing activity and high avidity and have the potential for developing an effective vaccine against HIV.