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HIV vaccine mediated efficacy, using an expanded live attenuated recombinant varicella virus-vectored SIV rSVV-SIVgag/env vaccine prime with adjuvanted SIV-Env and SIV-Gag protein boosts, was evaluated in a female rhesus macaques (RM) model against repeated intravaginal SIV challenges. Vaccination induced anti-SIV IgG responses and neutralizing antibodies were found in all vaccinated RMs. Three of the eight vaccinated RM remained uninfected (vaccinated and protected, VP) after 13 repeated challenges with the pathogenic SIVmac251-CX-1. The remaining five vaccinated and infected (VI) macaques had significantly reduced plasma viral loads compared with the infected controls (IC). A significant increase in systemic central memory CD4+ T cells and mucosal CD8+ effector memory T-cell responses was detected in vaccinated RMs compared to controls. Variability in lymph node SIV-Gag and Env specific CD4+ and CD8+ T cell cytokine responses were detected in the VI RMs while all three VP RMs had more durable cytokine responses following vaccination and prior to challenge. VI RMs demonstrated predominately SIV-specific monofunctional cytokine responses while the VP RMs generated polyfunctional cytokine responses. This study demonstrates that varicella virus-vectored SIV vaccination with protein boosts induces a 37.5% efficacy rate against pathogenic SIV challenge by generating mucosal memory, virus specific neutralizing antibodies, binding antibodies, and polyfunctional T-cell responses. [ABSTRACT FROM AUTHOR]

Broadly neutralizing antibodies (bNAbs) have remarkable breadth and potency against most HIV-1 subtypes and are able to prevent HIV-1 infection in animal models. However, bNAbs are extremely difficult to induce by vaccination. Defining the developmental pathways towards neutralization breadth can assist in the design of strategies to elicit protective bNAb responses by vaccination. Here, HIV-1 envelope glycoproteins (Env)-specific IgG+ B cells were isolated at various time points post infection from an HIV-1 infected elite neutralizer to obtain monoclonal antibodies (mAbs). Multiple antibody lineages were isolated targeting distinct epitopes on Env, including the gp120-gp41 interface, CD4-binding site, silent face and V3 region. The mAbs each neutralized a diverse set of HIV-1 strains from different clades indicating that the patient's remarkable serum breadth and potency might have been the result of a polyclonal mixture rather than a single bNAb lineage. High-resolution cryo-electron microscopy structures of the neutralizing mAbs (NAbs) in complex with an Env trimer generated from the same individual revealed that the NAbs used multiple strategies to neutralize the virus; blocking the receptor binding site, binding to HIV-1 Env N-linked glycans, and disassembly of the trimer. These results show that diverse NAbs can complement each other to achieve a broad and potent neutralizing serum response in HIV-1 infected individuals. Hence, the induction of combinations of moderately broad NAbs might be a viable vaccine strategy to protect against a wide range of circulating HIV-1 viruses. Author summary: Current behavioral strategies and (non-vaccine) biomedical interventions have been unable to stop the HIV-1/AIDS pandemic. A vaccine will be essential to eradicate the virus from the human population. The focus of HIV-1 vaccine design is to elicit broadly neutralizing antibodies that can block infection of viruses belonging to genetically diverse circulating HIV-1 clades. These bNAbs bind to the HIV-1 envelope glycoprotein (Env) and develop in a subset of HIV-1 infected individuals after several years of infection. Defining the developmental pathways and molecular determinants in env sequences that initiated bNAb responses in HIV-1 infected individuals can assist in the design of vaccines to elicit similar type of responses. Here, we isolated various antibody lineages from an HIV-1 infected individual and show that these antibodies target distinct epitopes on HIV-1 Env. None of the antibodies were extremely broad but we found that the antibody lineages each neutralized a different set of HIV-1 viruses from different clades. This suggests that the patient's remarkable serum breadth and potency may have been the result of a complementary polyclonal response. Vaccination strategies to induce combinations of moderately broad antibodies might be a viable approach to protect against infection. [ABSTRACT FROM AUTHOR]

A major goal of current HIV-1 vaccine design efforts is to induce broadly neutralizing antibodies (bNAbs). The VH1-2-derived bNAb IOMA directed to the CD4-binding site of the HIV-1 envelope glycoprotein is of interest because, unlike the better-known VH1-2-derived VRC01-class bNAbs, it does not require a rare short light chain complementarity-determining region 3 (CDRL3). Here, we describe three IOMA-class NAbs, ACS101-103, with up to 37% breadth, that share many characteristics with IOMA, including an average-length CDRL3. Cryo-electron microscopy revealed that ACS101 shares interactions with those observed with other VH1-2 and VH1-46-class bNAbs, but exhibits a unique binding mode to residues in loop D. Analysis of longitudinal sequences from the patient suggests that a transmitter/founder-virus lacking the N276 glycan might have initiated the development of these NAbs. Together these data strengthen the rationale for germline-targeting vaccination strategies to induce IOMA-class bNAbs and provide a wealth of sequence and structural information to support such strategies. HIV-1 vaccine design aims to induce broadly neutralizing antibodies such as IOMA, the only described antibody in its class. Here, the authors present the isolation, characterisation and structure of three additional antibodies within the IOMA class [ABSTRACT FROM AUTHOR]

Both SIV and SHIV are powerful tools for evaluating antibody-mediated prevention and treatment of HIV-1. However, owing to a lack of rhesus-derived SIV broadly neutralizing antibodies (bnAbs), testing of bnAbs for HIV-1 prevention or treatment has thus far been performed exclusively in the SHIV NHP model using bnAbs from HIV-1-infected individuals. Here we describe the isolation and characterization of multiple rhesus-derived SIV bnAbs capable of neutralizing most isolates of SIV. Eight antibodies belonging to two clonal families, ITS102 and ITS103 which target unique epitopes in the CD4bs region, were found to be broadly neutralizing and together neutralized all SIV strains tested. A rare feature of these bnAbs and two additional antibody families, ITS92 and ITS101 which mediate strain-specific neutralizing activity against SIV from sooty mangabeys (SIVsm), was their ability to achieve near complete (i.e. 100%) neutralization of moderately and highly neutralization-resistant SIV. Overall, these newly identified SIV bnAbs highlight the potential for evaluating HIV-1 prophylactic and therapeutic interventions using fully simian, rhesus-derived bnAbs in the SIV NHP model, thereby circumventing issues related to rapid antibody clearance of human-derived antibodies, Fc mismatch and limited genetic diversity of SHIV compared to SIV. Author summary: Antibodies that can bind to a virus and block infection are called neutralizing antibodies. In human immunodeficiency virus (HIV), antibodies that can block multiple strains of HIV circulating globally are known as broadly neutralizing antibodies. These broadly neutralizing antibodies have the potential to be used to treat HIV-infected individuals or to prevent HIV infection altogether. However, before these broadly neutralizing antibodies can safely be used in humans, they must first be tested in monkeys. Simian immunodeficiency virus (SIV), which causes an acquired immunodeficiency syndrome (AIDS)-like illness in Asian macaques that is similar to AIDS in humans, makes it possible to perform pre-clinical testing in monkeys but requires the use of antibodies that are specific to SIV. Another important consideration for testing antibodies in monkeys is that, like HIV, there are many different strains of SIV and some of these are highly resistant to neutralization. In this study we used blood from SIV-infected macaques to isolate several antibodies that can neutralize multiple strains of SIV, including strains that are typically resistant to neutralization. These SIV broadly neutralizing antibodies closely resemble HIV broadly neutralizing antibodies, making it possible now to test antibody-based vaccines or therapies for HIV using the SIV nonhuman primate model of AIDS. [ABSTRACT FROM AUTHOR]

Background: Recent studies have indicated that broadly neutralizing antibodies (bnAbs) in children may develop earlier after human immunodeficiency virus (HIV) infection compared to adults.Methods: We evaluated plasma from 212 antiretroviral therapy-naive children with HIV (1-3 years old). Neutralization breadth and potency was assessed using a panel of 10 viruses and compared to adults with chronic HIV. The magnitude, epitope specificity, and immunoglobulin (Ig)G subclass distribution of Env-specific antibodies were assessed using a binding antibody multiplex assay.Results: One-year-old children demonstrated neutralization breadth comparable to chronically infected adults, whereas 2- and 3-year-olds exhibited significantly greater neutralization breadth (P = .014). Likewise, binding antibody responses increased with age, with levels in 2- and 3-year-old children comparable to adults. Overall, there was no significant difference in antibody specificities or IgG subclass distribution between the pediatric and adult cohorts. It is interesting to note that the neutralization activity was mapped to a single epitope (CD4 binding site, V2 or V3 glycans) in only 5 of 38 pediatric broadly neutralizing samples, which suggests that most children may develop a polyclonal neutralization response.Conclusions: These results contribute to a growing body of evidence suggesting that initiating HIV immunization early in life may present advantages for the development of broadly neutralizing antibody responses. [ABSTRACT FROM AUTHOR]

Soluble HIV-1 envelope glycoprotein (Env) immunogens are a prime constituent of candidate vaccines designed to induce broadly neutralizing antibodies. Several lines of evidence suggest that enhancing Env immunogen thermostability can improve neutralizing antibody (NAb) responses. Here, we generated BG505 SOSIP.v9 trimers, which displayed virtually no reactivity with non-neutralizing antibodies and showed increased global and epitope thermostability, compared to previous BG505 SOSIP versions. Chemical crosslinking of BG505 SOSIP.v9 further increased the melting temperature to 91.3 °C, which is almost 25 °C higher than that of the prototype SOSIP.664 trimer. Next, we compared the immunogenicity of a palette of BG505-based SOSIP trimers with a gradient of thermostabilities in rabbits. We also included SOSIP.v9 proteins in which a strain-specific immunodominant epitope was masked by glycans to redirect the NAb response to other subdominant epitopes. We found that increased trimer thermostability correlated with increased potency and consistency of the autologous NAb response. Furthermore, glycan masking steered the NAb response to subdominant epitopes without decreasing the potency of the autologous NAb response. In summary, SOSIP.v9 trimers and their glycan masked versions represent an improved platform for HIV-1 Env based vaccination strategies. [ABSTRACT FROM AUTHOR]

Broadly neutralizing antibodies (bNAbs), known to mediate immune control of HIV-1 infection, only develop in a small subset of HIV-1 infected individuals. Despite being traditionally associated with patients with high viral loads, bNAbs have also been observed in therapy naïve HIV-1+ patients naturally controlling virus replication [Virus Controllers (VCs)]. Thus, dissecting the bNAb response in VCs will provide key information about what constitutes an effective humoral response to natural HIV-1 infection. In this study, we identified a polyclonal bNAb response to natural HIV-1 infection targeting CD4 binding site (CD4bs), V3-glycan, gp120-gp41 interface and membrane-proximal external region (MPER) epitopes on the HIV-1 envelope (Env). The polyclonal antiviral antibody (Ab) response also included antibody-dependent cellular phagocytosis of clade AE, B and C viruses, consistent with both the Fv and Fc domain contributing to function. Sequence analysis of envs from one of the VCs revealed features consistent with potential immune pressure and virus escape from V3-glycan targeting bNAbs. Epitope mapping of the polyclonal bNAb response in VCs with bNAb activity highlighted the presence of gp120-gp41 interface and CD4bs antibody classes with similar binding profiles to known potent bNAbs. Thus, these findings reveal the induction of a broad and polyfunctional humoral response in VCs in response to natural HIV-1 infection. [ABSTRACT FROM AUTHOR]

The development of an effective human immunodeficiency virus (HIV-1) vaccine is a high global health priority. Soluble native-like HIV-1 envelope glycoprotein trimers (Env), including those based on the SOSIP design, have shown promise as vaccine candidates by inducing neutralizing antibody responses against the autologous virus in animal models. However, to overcome HIV-1's extreme diversity a vaccine needs to induce broadly neutralizing antibodies (bNAbs). Such bNAbs can protect non-human primates (NHPs) and humans from infection. The prototypic BG505 SOSIP.664 immunogen is based on the BG505 env sequence isolated from an HIV-1-infected infant from Kenya who developed a bNAb response. Studying bNAb development during natural HIV-1 infection can inform vaccine design, however, it is unclear to what extent vaccine-induced antibody responses to Env are comparable to those induced by natural infection. Here, we compared Env antibody responses in BG505 SOSIP-immunized NHPs with those in BG505 SHIV-infected NHPs, by analyzing monoclonal antibodies (mAbs). We observed three major differences between BG505 SOSIP immunization and BG505 SHIV infection. First, SHIV infection resulted in more clonal expansion and less antibody diversity compared to SOSIP immunization, likely because of higher and/or prolonged antigenic stimulation and increased antigen diversity during infection. Second, while we retrieved comparatively fewer neutralizing mAbs (NAbs) from SOSIP-immunized animals, these NAbs targeted more diverse epitopes compared to NAbs from SHIV-infected animals. However, none of the NAbs, either elicited by vaccination or infection, showed any breadth. Finally, SOSIP immunization elicited antibodies against the base of the trimer, while infection did not, consistent with the base being placed onto the virus membrane in the latter setting. Together these data provide new insights into the antibody response against BG505 Env during infection and immunization and limitations that need to be overcome to induce better responses after vaccination. Author summary: A vaccine against HIV-1 would present a major breakthrough in the fight against HIV/AIDS. However, HIV-1 diversity, in particular in the envelope glycoproteins, proves a major hurdle for HIV-1 vaccine design. While broadly neutralizing antibodies develop to some degree in 20–30% of HIV-1-infected individuals and can protect non-human primates (NHPs) from virus infection, experimental HIV-1 vaccines have so far been unable to consistently induce such antibodies. A few years ago, soluble native-like HIV-1 envelope trimers, including SOSIP trimers, were developed which enabled the induction of neutralizing antibodies that could protect NHPs from infection with the sequence-matched virus. Here, we compared monoclonal antibodies from NHPs that were immunized with the SOSIP trimer or infected with a sequence-matched SHIV to better understand the successes and shortcomings of antibody development after SOSIP immunization compared to infection. Antibodies induced by infection were less diverse, but more clonally expanded and more potent in neutralizing the autologous virus. This is most likely a result of more and longer antigen stimulation and increased diversity of the envelope trimer during infection. Mimicking this extended antigen stimulation and variation with vaccination strategies might help to induce (broadly) neutralizing antibodies more efficiently. [ABSTRACT FROM AUTHOR]

A primary goal of HIV-1 vaccine development is the consistent elicitation of protective, neutralizing antibodies. While highly similar neutralizing antibodies (nAbs) have been isolated from multiple HIV-infected individuals, it is unclear whether vaccination can consistently elicit highly similar nAbs in genetically diverse primates. Here, we show in three outbred rhesus macaques that immunization with Env elicits a genotypically and phenotypically conserved nAb response. From these vaccinated macaques, we isolated four antibody lineages that had commonalities in immunoglobulin variable, diversity, and joining gene segment usage. Atomic-level structures of the antigen binding fragments of the two most similar antibodies showed nearly identical paratopes. The Env binding modes of each of the four vaccine-induced nAbs were distinct from previously known monoclonal HIV-1 neutralizing antibodies, but were nearly identical to each other. The similarities of these antibodies show that the immune system in outbred primates can respond to HIV-1 Env vaccination with a similar structural and genotypic solution for recognizing a particular neutralizing epitope. These results support rational vaccine design for HIV-1 that aims to reproducibly elicit, in genetically diverse primates, nAbs with specific paratope structures capable of binding conserved epitopes. Author summary: The primate immune system generates a diverse repertoire of pathogen-binding proteins called antibodies. Primate antibody repertoires must be diverse to respond to infection by many different pathogens. In this study, we characterized the structure and genetic features of HIV-1 inhibitory antibodies from vaccinated monkeys as a model for human vaccination. We found that individual monkeys responded to HIV vaccination by generating highly similar HIV-1 neutralizing antibodies. The antibodies had nearly identical structures and genetic makeups. Thus, the immune systems of different monkeys generate a common solution for inhibiting viral protein function and infectivity. Success of vaccines in multiple recipients may be augmented by the ability of immune systems to reproducibly devise a common antibody solution to attack vulnerable sites on pathogens. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Broadly neutralizing antibody (bnAb) induction is a high priority for effective HIV-1 vaccination. VRC01-class bnAbs that target the CD4 binding site (CD4bs) of trimeric HIV-1 envelope (Env) glycoprotein spikes are particularly attractive to elicit because of their extraordinary breadth and potency of neutralization in vitro and their ability to protect against infection in animal models. Glycans bordering the CD4bs impede the binding of germline-reverted forms of VRC01-class bnAbs and therefore constitute a barrier to early events in initiating the correct antibody lineages. Deleting a subset of these glycans permits Env antigen binding but not virus neutralization, suggesting that additional barriers impede germline-reverted VRC01-class antibody binding to functional Env trimers. We investigated the requirements for functional Env trimer engagement of VRC01-class naïve B cell receptors by using virus neutralization and germline-reverted antibodies as surrogates for the interaction. Targeted deletion of a subset of N-glycans bordering the CD4bs, combined with Man5 enrichment of remaining N-linked glycans that are otherwise processed into larger complex-type glycans, rendered HIV-1 426c Env-pseudotyped virus (subtype C, transmitted/founder) highly susceptible to neutralization by near germline forms of VRC01-class bnAbs. Neither glycan modification alone rendered the virus susceptible to neutralization. The potency of neutralization in some cases rivaled the potency of mature VRC01 against wildtype viruses. Neutralization by the germline-reverted antibodies was abrogated by the known VRC01 resistance mutation, D279K. These findings improve our understanding of the restrictions imposed by glycans in eliciting VRC01-class bnAbs and enable a neutralization-based strategy to monitor vaccine-elicited early precursors of this class of bnAbs. [ABSTRACT FROM AUTHOR]

The correct Funding statement is: The work is jointly funded by the Medical Research Council (MRC) UK and the UK Department for International Development (DFID) under the MRC/DFID Concordat agreements (G1001757 and MR/N023668/1); National Institutes of Health grant P01 AI100657; National Institutes of Health grant UM1-AI100645 to the Duke Center for HIV/AIDS Vaccine Immunology-Immunogen Discovery (CHAVI-ID) - subcontract 210782; National Institutes of Health contract HHSN27201100016C; the European Union's Horizon 2020 research and innovation programme under grant agreement No 681137; T.H. and Q.S. are the Jenner Institute Investigators, and Q.S. is a James Martin Senior fellow. Reference 1 Capucci S, Wee EG, Schiffner T, LaBranche CC, Borthwick N, Cupo A, et al. (2017) HIV-1-neutralizing antibody induced by simian adenovirus- and poxvirus MVA-vectored BG505 native-like envelope trimers. [Extracted from the article]

The article discusses on the efficacy of neutralizing antibody against cell-associated SHIV infection. It cites on the administration of neutralizing antibodies as an ideal property of an HIV-1 vaccine. The article also discusses on the establishment of intravenous cell-associated SHIV infection with the use of allogeneic cells.

Development of broadly neutralizing antibodies (bnAbs) against HIV-1 usually requires prolonged infection and induction of Abs with unusual features, such as long heavy-chain complementarity-determining region 3 (HCDR3) loops. Here we sought to determine whether the repertoires of HIV-1-naïve individuals contain Abs with long HCDR3 loops that could mediate HIV-1 neutralization. We interrogated at massive scale the structural properties of long Ab HCDR3 loops in HIV-1-naïve donors, searching for structured HCDR3s similar to those of the HIV-1 bnAb PG9. We determined the nucleotide sequences encoding 2.3 x 107 unique HCDR3 amino acid regions from 70 different HIV-1-naïve donors. Of the 26,917 HCDR3 loops with 30-amino acid length identified, we tested 30 for further study that were predicted to have PG9-like structure when chimerized onto PG9. Three of these 30 PG9 chimeras bound to the HIV-1 gp120 monomer, and two were neutralizing. In addition, we found 14 naturally occurring HCDR3 sequences that acquired the ability to bind to the HIV-1 gp120 monomer when adding 2- to 7-amino acid mutations via computational design. Of those 14 designed Abs, 8 neutralized HIV-1, with IC50 values ranging from 0.7 to 98 µg/mL. These data suggest that the repertoire of HIV- 1-naïve individuals contains rare B cells that encode HCDR3 loops that bind or neutralize HIV-1 when presented on a PG9 background with relatively few or no additional mutations. Long HCDR3 sequences are present in the HIV-naïve B-cell repertoire, suggesting that this class of bnAbs is a favorable target for rationally designed preventative vaccine efforts. [ABSTRACT FROM AUTHOR]

Intravascular delivery of broadly neutralizing antibodies (bnAbs) has shown promise for prevention and treatment of HIV infection. However, multiple IV administrations in geographic locations with poor accessibility to medical care have practical limitations. We have assessed the efficacy of plant-derived PGT121 delivered subcutaneously (SC) against pre-and post-intravaginal challenge using a rigorous SHIV-SF162P3 macaque protection model. SC administered PGT121 exhibited a longer serum half-life than IV administration and was more consistent than intramuscular delivery. A dose of 3.5mg/kg PGT121 prevented infection at a minimum ID50 neutralization titer of 1:295 while 5mg/kg protected five of six macaques when delivered immediately post-challenge. These results suggest the utility of plant-derived bnAbs delivered SC for HIV prevention. [ABSTRACT FROM AUTHOR]

The membrane proximal external region (MPER) of the gp41 subunit of the HIV-1 envelope glycoprotein (Env) contains determinants for broadly neutralizing antibodies and has remained an important focus of vaccine design. However, creating an immunogen that elicits broadly neutralizing antibodies to this region has proven difficult in part due to the relative inaccessibility of the MPER in the native conformation of Env. Here, we describe the antigenicity and immunogenicity of a panel of oligomeric gp41 immunogens designed to model a fusion-intermediate conformation of Env in order to enhance MPER exposure in a relevant conformation. The immunogens contain segments of the gp41 N- and C-heptad repeats to mimic a trapped intermediate, followed by the MPER, with variations that include different N-heptad lengths, insertion of extra epitopes, and varying C-termini. These well-characterized immunogens were evaluated in two different immunization protocols involving gp41 and gp140 proteins, gp41 and gp160 DNA primes, and different immunization schedules and adjuvants. We found that the immunogens designed to reduce extension of helical structure into the MPER elicited the highest MPER antibody binding titers, but these antibodies lacked neutralizing activity. The gp41 protein immunogens also elicited higher MPER titers than the gp140 protein immunogen. In prime-boost studies, the best MPER responses were seen in the groups that received DNA priming with gp41 vectors followed by gp41 protein boosts. Finally, although titers to the entire protein immunogen were similar in the two immunization protocols, MPER-specific titers differed, suggesting that the immunization route, schedule, dose, or adjuvant may differentially influence MPER immunogenicity. These findings inform the design of future MPER immunogens and immunization protocols. [ABSTRACT FROM AUTHOR]

Previously we described induction of cross-reactive HIV-1 neutralizing antibody responses in rabbits using a soluble HIV-1 gp140 envelope glycoprotein (Env) in an adjuvant containing monophosphoryl lipid A (MPL) and QS21 (AS02A). Here, we compared different forms of the same HIV-1 strain R2 Env for antigenic and biophysical characteristics, and in rabbits characterized the extent of B cell induction for specific antibody expression and secretion and neutralizing responses. The forms of this Env that were produced in and purified from stably transformed 293T cells included a primarily dimeric gp140, a trimeric gp140 appended to a GCN4 trimerization domain (gp140-GCN4), gp140-GCN4 with a 15 amino acid flexible linker between the gp120 and gp41 ectodomain (gp140-GCN4-L), also trimeric, and a gp140 with the flexible linker purified from cell culture supernatants as either dimer (gp140-L(D)) or monomer (gp140-L(M)). Multimeric states of the Env proteins were assessed by native gel electrophoresis and analytical ultracentrifugation. The different forms of gp140 bound broadly cross-reactive neutralizing (BCN) human monoclonal antibodies (mAbs) similarly in ELISA and immunoprecipitation assays. All Envs bound CD4i mAbs in the presence and absence of sCD4, as reported for the R2 Env. Weak neutralization of some strains of HIV-1 was seen after two additional doses in AS02A. Rabbits that were given a seventh dose of gp140-GCN4-L developed BCN responses that were weak to moderate, similar to our previous report. The specificity of these responses did not appear similar to that of any of the known BCN human mAbs. Induction of spleen B cell and plasma cells producing immunoglobulins that bound trimeric gp140-GCN4-L was vigorous, based on ELISpot and flow cytometry analyses. The results demonstrate that highly purified gp140-GCN4-L trimer in adjuvant elicits BCN responses in rabbits accompanied by vigorous B cell induction. [ABSTRACT FROM AUTHOR]

The induction of broadly reacting neutralizing antibodies has been a major goal of HIV vaccine research. Characterization of a pathogenic CCR5 (R5)-tropic SIV/HIV chimeric virus (SHIV) molecular clone (SHIVAD8-EO) revealed that eight of eight infected animals developed cross-reactive neutralizing antibodies (NAbs) directed against an envelope glycoprotein derived from the heterologous HIV-1DH12 strain. A panel of plasmas, collected from monkeys inoculated with either molecularly cloned or uncloned SHIVAD8 stocks, exhibited cross-neutralization against multiple tier 1 and tier 2 HIV-1 clade B isolates. One SHIVAD8-infected animal also developed NAbs against clades A and C HIV-1 strains. In this particular infected macaque, the cross-reacting anti–HIV-1 NAbs produced between weeks 7 and 13 were directed against a neutralization-sensitive virus strain, whereas neutralizing activities emerging at weeks 41–51 targeted more neutralization-resistant HIV-1 isolates. These results indicate that the SHIVAD8 macaque model represents a potentially valuable experimental system for investigating B-cell maturation and the induction of cross-reactive NAbs directed against multiple HIV-1 strains. [ABSTRACT FROM AUTHOR]

The following information is missing from the Funding statement: M.J.B. and A.H. received funding from the Intramural Research Program of the NIH; National Institute of Environmental Health Sciences (ZIC ES103326 to M.J.B). The correct statement is: The cryo-EM data of CH235 UCA bound to Man5-enriched CH505.N279K.G458Y.SOSIP.664 have been deposited to EMDB database with the accession code EMD-20735, and the fitted coordinates have been deposited to the RCSB database with accession number 6UDA. [Extracted from the article]

Immune complex (IC) vaccines have been successfully used to increase immune responses against various pathogens, including HIV-1. Additionally, IC vaccines can induce qualitatively different antibody responses, with distinct antigenic specificities compared to the same antigens used alone. Here we measured the HIV-1-specific antibody responses in female New Zealand White rabbits after immunization with ICs made from BG505 SOSIP.664 trimers (BG505 trimers) and three rabbit monoclonal antibodies (MAbs) with different neutralization profiles. Two of the MAbs were specific for a hole in the glycan shield of the BG505 trimer, while the third, which bound less avidly, was specific for determinants at the gp41-gp120 interface. We found that immunization with one of the glycan-hole-specific ICs resulted in lower levels of trimer-binding antibodies compared to vaccination with the uncomplexed trimer, and that ICs made using either of the glycan-hole-specific MAbs resulted in lower rates of anti-trimer antibody decay. We concluded that ICs based on MAbs that bound to the immunodominant glycan hole epitope likely diverted antibody responses, to some extent, away from this site and to other regions of the trimer. However, this outcome was not accompanied by a widening of the breadth or an increase in the potency of neutralizing antibody responses compared with uncomplexed trimers. [ABSTRACT FROM AUTHOR]

Background:There is a well-acknowledged need for an effective AIDS vaccine that protects against HIV-1 infection or limits invivo viral replication. The objective of these studies is to develop a replication-competent, vaccine vector based on the adenovirus serotype 4 (Ad4) virus expressing HIV-1 envelope (Env) 1086 clade C glycoprotein. Ad4 recombinant vectors expressing Env gp160 (Ad4Env160), Env gp140 (Ad4Env140), and Env gp120 (Ad4Env120) were evaluated. Methods:The recombinant Ad4 vectors were generated with a full deletion of the E3 region of Ad4 to accommodate the env gene sequences. The vaccine candidates were assessed in vitro following infection of A549 cells for Env-specific protein expression and for posttranslational transport to the cell surface as monitored by the binding of broadly neutralizing antibodies (bNAbs). The capacity of the Ad4Env vaccines to induce humoral immunity was evaluated in rabbits for Env gp140 and V1V2-specific binding antibodies, and HIV-1 pseudovirus neutralization. Mice immunized with the Ad4Env160 vaccine were assessed for IFNγ T cell responses specific for overlapping Env peptide sets. Results:Robust Env protein expression was confirmed by western blot analysis and recognition of cell surface Env gp160 by multiple bNAbs. Ad4Env vaccines induced humoral immune responses in rabbits that recognized Env 1086 gp140 and V1V2 polypeptide sequences derived from 1086 clade C, A244 clade AE, and gp70 V1V2 CASE A2 clade B fusion protein. The immune sera efficiently neutralized tier 1 clade C pseudovirus MW965.26 and neutralized the homologous and heterologous tier 2 pseudoviruses to a lesser extent. Env-specific T cell responses were also induced in mice following Ad4Env160 vector immunization. Conclusions:The Ad4Env vaccine vectors express high levels of Env glycoprotein and induce both Env-specific humoral and cellular immunity thus supporting further development of this new Ad4 HIV-1 Env vaccine platform in Phase 1 clinical trials. [ABSTRACT FROM AUTHOR]

Sensitive assays are needed to meaningfully assess low levels of neutralizing antibodies (NAbs) that may be important for protection against the acquisition of HIV-1 infection in vaccine recipients. The current assay of choice uses a non-lymphoid cell line (TZM-bl) that may lack sensitivity owing to over expression of CD4 and CCR5. We used transfection of a human CD4+/CXCR4+/α4β7+ T-lymphoblastoid cell line (A3.01) with a CMV IE promoter-driven CCR5neo vector to stably express CCR5. The resulting line, designated A3R5, is permissive to a wide range of CCR5-tropic circulating strains of HIV-1, including HIV-1 molecular clones containing a Tat-inducible Renilla luciferase reporter gene and expressing multiple Env subtypes. Flow cytometric analysis found CCR5 surface expression on A3R5 cells to be markedly less than TZM-bl but similar to CD3.8 stimulated PBMC. More importantly, neutralization mediated by a diverse panel of monoclonal antibodies, HIV-1 positive polyclonal sera and sCD4 was consistently greater in A3R5 compared to TZM-bl cells. The A3R5 cell line provides a novel approach to guide the development and qualification of promising new HIV-1 vaccine immunogens. [ABSTRACT FROM AUTHOR]

Newcastle disease virus (NDV) expressing HIV-1 BaL gp160 was evaluated either alone or with monomeric BaL gp120 and BaL SOSIP gp140 protein in a prime-boost combination in guinea pigs to enhance envelope (Env)-specific humoral and mucosal immune responses. We showed that a regimen consisting of an NDV prime followed by a protein boost elicited stronger serum and mucosal Th-1-biased IgG responses and neutralizing antibody responses than NDV-only immunizations. Additionally, these responses were higher after the gp120 than after the SOSIP gp140 protein boost. [ABSTRACT FROM AUTHOR]

The role of vaccine-induced anti-V2 Abs was tested in three protection experiments in rhesus macaques. In an experiment using immunogens similar to those in the RV144 vaccine trial (Anti-envelope [Env]), nine rhesus macaques were coimmunized with gp16092TH023 DNA and SIV gag and gp120A244 and gp120MN proteins. In two V2-focused experiments (Anti-V2 and Anti-V2 Mucosal), nine macaques in each group were immunized with V1V292TH023 DNA, V1V2A244 and V1V2CasaeA2 proteins, and cyclic V2CaseA2 peptide. DNA and protein immunogens, formulated in Adjuplex, were given at 0, 4, 12, and 20 weeks, followed by intrarectal SHIVBaL.P4 challenges. Peak plasma viral loads (PVL) of 106-107 copies/ml developed in all nine sham controls. Overall, PVL was undetectable in one third of immunized macaques, and two animals tightly controlled the virus with the Anti-V2 Mucosal vaccine strategy. In the Anti-Env study, Abs that captured or neutralized SHIVBaL.P4 inversely correlated with PVL. Conversely, no correlation with PVL was found in the Anti-V2 experiments with nonneutralizing plasma Abs that only captured virus weakly. Titers of Abs against eight V1V2 scaffolds and cyclic V2 peptides were comparable between controllers and noncontrollers as were Ab-dependent cellular cytotoxicity and Ab-dependent cell-mediated virus inhibition activities against SHIV-infected target cells and phagocytosis of gp120-coated beads. The Anti-Env experiment supports the role of vaccineelicited neutralizing and nonneutralizing Abs in control of PVL. However, the two V2-focused experiments did not support a role for nonneutralizing V2 Abs alone in controlling PVL, as neither Ab-dependent cellular cytotoxicity, Ab-dependent cell-mediated virus inhibition, nor phagocytosis correlated inversely with heterologous SHIVBaL.P4 infection. [ABSTRACT FROM AUTHOR]

The induction of broadly neutralizing antibodies (bNAbs) is a major goal in vaccine research. HIV-1-infected individuals that develop exceptionally strong bNAb responses, termed elite neutralizers, can inform vaccine design by providing blueprints for the induction of similar bNAb responses. We describe a new recombinant native-like envelope glycoprotein (Env) SOSIP trimer, termed AMC009, based on the viral founder sequences of an elite neutralizer. The subtype B AMC009 SOSIP protein formed stable native-like trimers that displayed multiple bNAb epitopes. Overall, its structure at 4.3-Å resolution was similar to that of BG505 SOSIP.664. The AMC009 trimer resembled one from a second elite neutralizer, AMC011, in having a dense and complete glycan shield. When tested as immunogens in rabbits, the AMC009 trimers did not induce autologous neutralizing antibody (NAb) responses efficiently while the AMC011 trimers did so very weakly, outcomes that may reflect the completeness of their glycan shields. The AMC011 trimer induced antibodies that occasionally cross-neutralized heterologous tier 2 viruses, sometimes at high titer. Cross-neutralizing antibodies were more frequently elicited by a trivalent combination of AMC008, AMC009, and AMC011 trimers, all derived from subtype B viruses. Each of these three individual trimers could deplete the NAb activity from the rabbit sera. Mapping the polyclonal sera by electron microscopy revealed that antibodies of multiple specificities could bind to sites on both autologous and heterologous trimers. These results advance our understanding of how to use Env trimers in multivalent vaccination regimens and the immunogenicity of trimers derived from elite neutralizers. [ABSTRACT FROM AUTHOR]

The development of broadly neutralizing antibodies (BNAbs) in HIV infection is a result of long-term coevolutionary interaction between viruses and antibodies. Understanding how this interaction promotes the increase of neutralization breadth during infection will improve the way in which AIDS vaccine strategies are designed. In this paper, we used SIV-infected rhesus macaques as a model to study the development of neutralization breadth by infecting rhesus macaques with longitudinal NAb escape variants and evaluating the kinetics of NAb response and viral evolution. We found that the infected macaques developed a stepwise NAb response against escape variants and increased neutralization breadth during the course of infection. Furthermore, the increase of neutralization breadth correlated with the duration of infection but was independent of properties of the inoculum, viral loads, or viral diversity during infection. These results imply that the duration of infection was the main factor driving the development of BNAbs. These data suggest the importance of novel immunization strategies to induce effective NAb response against HIV infection by mimicking long-term infection. [ABSTRACT FROM AUTHOR]

The correlation of HIV-specific antibody-dependent cellular cytotoxicity (ADCC) responses with protection from and delayed progression of HIV-1 infection provides a rationale to leverage ADCC-mediating antibodies for treatment purposes. We evaluated ADCC mediated by different combinations of 2 to 6 neutralizing and non-neutralizing anti-HIV-1 Envelope (Env) mAbs, using concentrations ≤ 1 μg/mL, to identify combinations effective at targeting latent reservoir HIV-1 viruses from 10 individuals. We found that within 2 hours, combinations of 3 mAbs mediated more than 30% killing of HIV-infected primary CD4+ T cells in the presence of autologous NK cells, with the combination of A32 (C1C2), DH511.2K3 (MPER), and PGT121 (V3) mAbs being the most effective. Increasing the incubation of target and effector cells in the presence of mAb combinations from 2 to 24 hours resulted in increased specific killing of infected cells, even with neutralization-resistant viruses. The same combination eliminated reactivated latently HIV-1-infected cells in an ex vivo quantitative viral outgrowth assay. Therefore, administration of a combination of 3 mAbs should be considered in planning in vivo studies seeking to eliminate persistently HIV-1-infected cells. [ABSTRACT FROM AUTHOR]

We covalently attached human immunodeficiency virus type 1 (HIV-1) Env SOSIP trimers to iron oxide nanoparticles (IO-NPs) to create a particulate immunogen for neutralizing antibody (NAb) induction. The attached trimers, ∼20 per particle, retained native-like antigenicity, judged by reactivity with NAbs and non-NAbs. Bivalent (BG505 and B41) trimer IO-NPs were made, as were IO-NPs displaying B41 trimers carrying a PADRE T-cell helper epitope (TCHE). We immunized mice with B41 soluble or IO-NP trimers after PADRE peptide priming. After two immunizations, IO-NP presentation and the TCHE tag independently and substantially increased anti-trimer antibody responses, but titer differences waned after two further doses. Notable and unexpected findings were that autologous NAbs to the N289 glycan hole epitope were consistently induced in mice given soluble but not IO-NP trimers. Various recombinant mannose binding lectins (MBLs) and MBLs in sera of both murine and human origin bound to soluble and IO-NP trimers. MBL binding occluded the autologous NAb epitope on the B41 IO-NP trimers, which may contribute to its poor immunogenicity. The exposure of a subset of broadly active NAb epitopes was also impaired by MBL binding, which could have substantial implications for the utility of trimer-bearing nanoparticles in general and perhaps also for soluble Env proteins. [ABSTRACT FROM AUTHOR]

ABSTRACT Recombinant soluble HIV-1 envelope glycoprotein (Env) SOSIP trimers are a design platform for inducing broadly neutralizing antibodies (bNAbs) by vaccination. To date, these and alternative designs of native-like trimers, given singly or in pairs, have not induced bNAbs in test animals such as rabbits or macaques. Here, we have evaluated whether trivalent and tetravalent combinations of SOSIP trimers from clades A, B, and C, delivered simultaneously or sequentially, induce better neutralizing antibody responses in rabbits than when given alone. None of the tested formulations led to the induction of bNAbs. We found that BG505 clade A trimers dominated the autologous NAb responses induced by combinations, which probably relates to the presence of immunodominant glycan holes on the BG505 trimer. Furthermore, autologous NAb responses to all individual trimers were reduced when they were delivered in combinations compared with when delivered alone, suggesting that immunogen interference had occurred. Finally, in a sequential regimen, a heterologous clade C trimer cross-boosted NAb responses that were primed by earlier immunizations with clade A and B trimers. Taken together, these findings should allow us to improve the design of immunization regimens based on native-like HIV-1 Env trimers. IMPORTANCE: A successful HIV-1 vaccine most probably requires a trimeric envelope glycoprotein (Env) component, as Env is the only viral protein on the surface of the virus and therefore the only target for neutralizing antibodies. Native-like Env trimers can induce strain-specific neutralizing antibodies but not yet broadly neutralizing antibodies. To try to broaden the antibody response, we immunized rabbits with soluble native-like Env trimers from three different clades using monovalent, multivalent, and sequential regimens. We found that the neutralizing antibody response against each immunogen was reduced when the immunogens were delivered in combination or sequentially compared to the monovalent regimen. In contrast, when the Env trimers from different clades were delivered sequentially, the neutralizing antibody response could be crossboosted. Although the combination of native-like Env trimers from different clades did not induce broadly neutralizing antibodies, the results provide clues on how to use native-like trimers in vaccination experiments. [ABSTRACT FROM AUTHOR]

To provide protective immunity against circulating primary HIV-1 strains, a vaccine most likely has to induce broadly neutralizing antibodies to the HIV-1 envelope glycoprotein (Env) spike. Recombinant Env trimers such as the prototype BG505 SOSIP.664 that closely mimic the native Env spike can induce autologous neutralizing antibodies (NAbs) against relatively resistant (tier 2) primary viruses. Ideally, Env immunogens should present broadly neutralizing antibody epitopes but limit the presentation of immunodominant non-NAb epitopes that might induce off-target and potentially interfering responses. The V3 loop in gp120 is such a non-NAb epitope that can effectively elicit non-NAbs when animals are immunized with SOSIP.664 trimers. V3 immunogenicity can be diminished, but not abolished, by reducing the conformational flexibility of trimers via targeted sequence changes, including an A316W substitution in V3, that create the SOSIP.v4.1 and SOSIP.v5.2 variants. Here, we further modified these trimer designs by introducing leucine residues at V3 positions 306 and 308 to create hydrophobic interactions with the tryptophan residue at position 316 and with other topologically proximal sites in the V1V2 domain. Together, these modifications further stabilized the resulting SOSIP.v5.2 S306L/R308L trimers in the prefusion state in which V3 is sequestered. When we tested these trimers as immunogens in rabbits, the induction of V3 non-NAbs was significantly reduced compared with the SOSIP.v5.2 trimers and even more so compared with the SOSIP.664 prototype, without affecting the autologous NAb response. Hence, these additional trimer sequence modifications may be beneficial for immunization strategies that seek to minimize off-target non- NAb responses. [ABSTRACT FROM AUTHOR]

In the search for effective immunologic interventions to prevent and treat HIV-1 infection, standardized reference reagents are a cost-effective way to maintain robustness and reproducibility among immunological assays. To support planned and ongoing studies where clade C predominates, here we describe three virus panels, chosen from 200 well-characterized clade C envelope (Env)-pseudotyped viruses from early infection. All 200 Envs were expressed as a single round of replication pseudoviruses and were tested to quantify neutralization titers by 16 broadly neutralizing antibodies (bnAbs) and sera from 30 subjects with chronic clade C infections. We selected large panels of 50 and 100 Envs either to characterize cross-reactive breadth for sera identified as having potent neutralization activity based on initial screening or to evaluate neutralization magnitude-breadth distributions of newly isolated antibodies. We identified these panels by downselection after hierarchical clustering of bnAb neutralization titers. The resulting panels represent the diversity of neutralization profiles throughout the range of virus sensitivities identified in the original panel of 200 viruses. A small 12-Env panel was chosen to screen sera from vaccine trials or naturalinfection studies for neutralization responses. We considered panels selected by previously described methods but favored a computationally informed method that enabled selection of viruses representing diverse neutralization sensitivity patterns, given that we do not a priori know what the neutralization-response profile of vaccine sera will be relative to that of sera from infected individuals. The resulting 12- Env panel complements existing panels. Use of standardized panels enables direct comparisons of data from different trials and study sites testing HIV-1 clade C-specific products. IMPORTANCE HIV-1 group M includes nine clades and many recombinants. Clade C is the most common lineage, responsible for roughly half of current HIV-1 infections and is a focus for vaccine design and testing. Standard reference reagents, particularly virus panels to study neutralization by antibodies, are crucial for developing cost-effective and yet rigorous and reproducible assays against diverse examples of this variable virus. We developed clade C-specific panels for use as standardized reagents to monitor complex polyclonal sera for neutralization activity and to characterize the potency and breadth of cross-reactive neutralization by monoclonal antibodies, whether engineered or isolated from infected individuals. We chose from 200 southern African, clade C envelope-pseudotyped viruses with neutralization ti- ters against 16 broadly neutralizing antibodies and 30 sera from chronic clade C infections. We selected panels to represent the diversity of bnAb neutralization profiles and Env neutralization sensitivities. Use of standard virus panels can facilitate comparison of results across studies and sites. [ABSTRACT FROM AUTHOR]

Native-like trimers of the SOSIP design are being developed as immunogens in human immunodeficiency virus type 1 (HIV-1) vaccine development programs. These trimers display the epitopes for multiple broadly neutralizing antibodies (bNAbs) but can also expose binding sites for some types of nonneutralizing antibodies (non-NAbs). Among the latter are epitopes in the gp120 V3 region that are highly immunogenic when SOSIP trimers are evaluated in animal models. It is presently uncertain whether antibodies against V3 can interfere with the induction of NAbs, but there are good arguments in favor of suppressing such "off-target" immune responses. Accordingly, we have assessed how to minimize the exposure of V3 non-NAb epitopes and thereby reduce their immunogenicity by introducing N-glycans within the V3 region of BG505 SOSIP trimers. We found that inserting glycans at positions 306 and 314 (termed M1 and M7) markedly reduced V3 antigenicity while improving the presentation of trimer apex bNAb epitopes. Both added glycans were shown to be predominantly of the Man6GlcNAc2 form. The additional introduction of the E64K ground-state stabilizing substitution markedly reduced or ablated soluble CD4 (sCD4) induction of non-NAb epitopes in V3 and/or associated with the coreceptor binding site. When a V3 glycan- and E64K-modified trimer variant, BG505 SOSIP.664-E64K.M1M7, was tested in rabbits, V3 immunogenicity was eliminated while the autologous NAb response was unchanged. [ABSTRACT FROM AUTHOR]

A major goal for HIV-1 vaccine development is an ability to elicit strong and durable broadly neutralizing antibody (bNAb) responses. The trimeric envelope glycoprotein (Env) spikes on HIV-1 are known to contain multiple epitopes that are susceptible to bNAbs isolated from infected individuals. Nonetheless, all trimeric and monomeric Env immunogens designed to date have failed to elicit such antibodies. We report the structure-guided design of HIV-1 cyclically permuted gp120 that forms homogeneous, stable trimers, and displays enhanced binding to multiple bNAbs, including VRC01, VRC03, VRCPG04, PGT128, and the quaternary epitope-specific bNAbs PGT145 and PGDM1400. Constructs that were cyclically permuted in the V1 loop region and contained an N-terminal trimerization domain to stabilize V1V2-mediated quaternary interactions, showed the highest homogeneity and the best antigenic characteristics. In guinea pigs, a DNA prime-protein boost regimen with these new gp120 trimer immunogens elicited potent neutralizing antibody responses against highly sensitive Tier 1A isolates and weaker neutralizing antibody responses with an average titer of about 115 against a panel of heterologous Tier 2 isolates. A modest fraction of the Tier 2 virus neutralizing activity appeared to target the CD4 binding site on gp120. These results suggest that cyclically permuted HIV-1 gp120 trimers represent a viable platform in which further modifications may be made to eventually achieve protective bNAb responses. [ABSTRACT FROM AUTHOR]

Human Immunodeficiency Virus (HIV) Virus-Like Particles (VLPs) composed of HIV IIIB Gag and HIV BaL gp120/gp41 envelope are a pseudovirion vaccine capable of presenting antigens in their native conformations. To enhance the immunogenicity of the HIV Env antigen, VLPs were coupled to VesiVax Conjugatable Adjuvant Lipid Vesicles (CALV) containing one of four toll-like-receptor (TLR) ligands, each activating a receptor with distinct cellular localization and downstream pathways. C57BL/6 mice were vaccinated by intranasal prime followed by two sub-cheek boosts and their sera immunoglobulin and neutralizing potency were measured over a duration of 3 months after vaccination. PBS control, VLPs alone, CALV + VLPs, and VLPs complexed with CALV and ligands for TLR2 (PAM3CAG), TLR3 (dsRNA), TLR4 (MPLA), or TLR7/8 (resiquimod) were evaluated based on antibody titer, IgG1 and IgG2c class switching, germinal center formation, T follicular cells and potency of neutralizing antibodies. Consistently, the TLR3 ligand dsRNA complexed to CALV and in combination with VLPs (CALV(dsRNA) + VLPs) induced the strongest response. CALV(dsRNA) + VLPs induced the highest titers against the recombinant vaccine antigens clade B Bal gp120 and pr55 Gag. Additionally, CALV(dsRNA) + VLPs induced cross-clade antibodies, represented by high titers of antibody to clade c 96ZM651 gp120. CALV(dsRNA) + VLPs induced predominantly IgG2c over IgG1, a response associated with T helper type 1 (Th1)-like cytokines. In turn, CALV(dsRNA) + VLP immunized mice generated the most potent neutralizing antibodies against HIV strain MN.3. Finally, at time of sacrifice, a significant increase in germinal center B cells and T follicular cells was detected in mice which received CALV(dsRNA) + VLPs compared to PBS. Our results indicate that CALV(dsRNA) is a superior adjuvant for HIV VLPs in generating a Th1-like immunoglobulin profile, while prolonging lymph node germinal centers, T follicular cells and generating neutralizing antibodies to a highly sensitive tier 1A variant of HIV. [ABSTRACT FROM AUTHOR]

Enhancement of HIV-specific immunity is likely required to eliminate latent HIV infection. Here, we have developed an immunotherapeutic modality aimed to improve T cell-mediated clearance of HIV-1-infected cells. Specifically, we employed Dual-Affinity Re-Targeting (DART) proteins, which are bispecific, antibody-based molecules that can bind 2 distinct cell-surface molecules simultaneously. We designed DARTs with a monovalent HIV-1 envelope-binding (Env-binding) arm that was derived from broadly binding, antibody-dependent cellular cytotoxicity-mediating antibodies known to bind to HIV-infected target cells coupled to a monovalent CD3 binding arm designed to engage cytolytic effector T cells (referred to as HIVxCD3 DARTs). Thus, these DARTs redirected polyclonal T cells to specifically engage with and kill Env-expressing cells, including CD4+ T cells infected with different HIV-1 subtypes, thereby obviating the requirement for HIV-specific immunity. Using lymphocytes from patients on suppressive antiretroviral therapy (ART), we demonstrated that DARTs mediate CD8+ T cell clearance of CD4+ T cells that are superinfected with the HIV-1 strain JR-CSF or infected with autologous reservoir viruses isolated from HIV-infected-patient resting CD4+ T cells. Moreover, DARTs mediated CD8+ T cell clearance of HIV from resting CD4+ T cell cultures following induction of latent virus expression. Combined with HIV latency reversing agents, HIVxCD3 DARTs have the potential to be effective immunotherapeutic agents to clear latent HIV-1 reservoirs in HIV-infected individuals. [ABSTRACT FROM AUTHOR]

Several HIV envelope-targeting (Env-targeting) antibodies with broad and potent neutralizing activity have been identified and shown to have unusual features. Of these, the PG9 antibody has a long heavy chain complementarity determining region 3 (HCDR3) and possesses unique structural elements that interact with protein and glycan features of the HIV Env glycoprotein. Here, we used the Rosetta software suite to design variants of the PG9 antibody HCDR3 loop with the goal of identifying variants with increased potency and breadth of neutralization for diverse HIV strains. One variant, designated PG9_N100FY, possessed increased potency and was able to neutralize a diverse set of PG9-resistant HIV strains, including those lacking the Env N160 glycan, which is critical for PG9 binding. An atomic resolution structure of the PG9_N100FY fragment antigen binding (Fab) confirmed that the mutated residue retains the paratope surface when compared with WT PG9. Differential scanning calorimetry experiments revealed that the mutation caused a modest increase in thermodynamic stability of the Fab, a feature predicted by the computational model. Our findings suggest that thermodynamic stabilization of the long HCDR3 in its active conformation is responsible for the increased potency of PG9_N100FY, and strategies aimed at stabilizing this region in other HIV antibodies could become an important approach to in silico optimization of antibodies. [ABSTRACT FROM AUTHOR]

The membrane-proximal external region (MPER) of the human immunodeficiency virus, type 1 (HIV-1) envelope glycoprotein subunit gp41 is targeted by potent broadly neutralizing antibodies 2F5, 4E10, and 10E8. These antibodies recognize linear epitopes and have been suggested to target the fusion intermediate conformation of gp41 that bridges viral and cellular membranes. Anti-MPER antibodies exert different degrees of membrane interaction, which is considered to be the limiting factor for the generation of such antibodies by immunization. Here we characterize a fusion intermediate conformation of gp41 (gp41int-Cys) and show that it folds into an elongated~12- nm-long extended structure based on small angle x-ray scattering data. Gp41int-Cys was covalently linked to liposomes via its C-terminal cysteine and used as immunogen. The gp41int-Cys proteoliposomes were administered alone or in prime-boost regimen with trimeric envelope gp140CA018 in guinea pigs and elicited high anti-gp41 IgG titers. The sera interacted with a peptide spanning the MPER region, demonstrated competition with broadly neutralizing antibodies 2F5 and 4E10, and exerted modest lipid binding, indicating the presence of MPER-specific antibodies. Although the neutralization potency generated solely by gp140CA018 was higher than that induced by gp41int- Cys, the majority of animals immunized with gp41int-Cys proteoliposomes induced modest breadth and potency in neutralizing tier 1 pseudoviruses and replication-competent simian/ human immunodeficiency viruses in the TZM-bl assay as well as responses against tier 2 HIV-1 in the A3R5 neutralization assay. Our data thus demonstrate that liposomal gp41 MPER formulation can induce neutralization activity, and the strategy serves to improve breadth and potency of such antibodies by improved vaccination protocols. [ABSTRACT FROM AUTHOR]

One strategy being evaluated for HIV-1 vaccine development is focusing immune responses towards neutralizing epitopes on the gp120 outer domain (OD) by removing the immunodominant, but non-neutralizing, inner domain. Previous OD constructs have not elicited strong neutralizing antibodies (nAbs). We constructed two immunogens, a monomeric gp120-OD and a trimeric gp120-ODx3, based on an M group consensus sequence (MCON6). Their biochemical and immunological properties were compared with intact gp120. Results indicated better preservation of critical neutralizing epitopes on gp120-ODx3. In contrast to previous studies, our immunogens induced potent, cross-reactive nAbs in rabbits. Although nAbs primarily targeted Tier 1 viruses, they exhibited significant breadth. Epitope mapping analyses indicated that nAbs primarily targeted conserved V3 loop elements. Although the potency and breadth of nAbs were similar for all three immunogens, nAb induction kinetics indicated that gp120-ODx3 was superior to gp120-OD, suggesting that gp120-ODx3 is a promising prototype for further gp120 OD-based immunogen development. [ABSTRACT FROM AUTHOR]

As an alternative to targeting HIV, we have developed vaccines targeting CCR5, a self-protein critically involved in HIV replication and pathogenesis. By displaying peptides derived from CCR5 at high density on the surface of virus-like particles, we can efficiently induce high-titer IgG antibodies against this self-molecule. Here, we investigated if prophylactic immunization of rhesus macaques with a particle-based vaccine targeting two regions of macaque CCR5 could prevent or suppress vaginal infection with highly virulent SIVmac251. Twelve macaques were vaccinated with a bacteriophage Qβ-based vaccine targeting macaque CCR5 (Qβ.CCR5). Six control animals were immunized with the Qβ platform alone. All animals immunized with Qβ.CCR5 developed high-titer anti-CCR5 antibody responses. Macaques were vaginally challenged with a high dose of SIVmac251. The mean peak viral RNA levels in the vaccinated groups were 30-fold lower than in the control group (106.8 versus 10803 copies/ml plasma). Three of the 12 vaccinated macaques dramatically suppressed SIV replication: peak viral loads were low (10³--104 RNA copies/ml), and SIV RNA became undetectable from six weeks onward. No viral RNA or DNA could be detected in colon and lymph node biopsies collected 13 months after challenge. In vivo depletion of CD8+ cells failed to induce a viral rebound. However, once anti-CCR5 antibody responses had waned, the 3 animals became infected after intravaginal and/or intravenous rechallenge. In conclusion, vaccination against CCR5 was associated with dramatic suppression of virus replication in a subset (25%) of macaques. These data support further research of vaccination against CCR5 to combat HIV infection [ABSTRACT FROM AUTHOR]

Broadly neutralizing antibodies (bNAbs) PG9 and PG16 were isolated from an International AIDS Vaccine Initiative (IAVI) Protocol G subject infected with human immunodeficiency virus type 1 (HIV-1) clade A. Both antibodies are highly potent and neutralize greater than 70% of viruses tested. We sought to begin immunogen design based on viral sequences from this patient; however, pseudoviruses prepared with 19 envelope sequences from this subject were resistant to neutralization by PG9 and PG16. Therefore, we used a bioinformatics approach to identify closely related viruses that were potentially sensitive to PG9 and PG16. A most-recent common ancestor (MRCA) sequence for the viral envelope (Env) was determined and aligned with 99 sub-type A gpl60 sequences from the Los Alamos HIV database. Virus BG505.W6M.ENV.C2 (BG505) was found to have the highest degree of homology (73%) to the MRCA sequence. Pseudoviruses prepared with this Env were sensitive to neutralization with a broad panel of bNAbs, including PG9 and PG16. When expressed by 293T cells as soluble gpl20, the BG505 monomer bound well to both PG9 and PG16. We further showed that a point mutation (LI 11 A) enabled more efficient production of a stable gpl20 monomer that preserves the major neutralization epitopes. Finally, we showed that an adjuvanted formulation of this gpl20 protein elicited neutralizing antibodies in rabbits (following a gpl20 DNA vaccine prime) and that the antisera competed with bNAbs from 3 classes of nonoverlapping epitopes. Thus, the BG505 Env protein warrants further investigation as an HIV vaccine candidate, as a stand-alone protein, or as a component of a vaccine vector. [ABSTRACT FROM AUTHOR]

The recent identification of human monoclonal antibodies with broad and potent neutralizing activity against HIV-1 (bnAbs) has resulted in substantial efforts to develop these molecules for clinical use in the prevention and treatment of HIV-1 infection. As with any protein therapeutic drug product, it is imperative to have qualified assays that can accurately detect and quantify anti-drug antibodies (ADA) that may develop in patients receiving passive administration of HIV-1 bnAbs. Here, we have optimized and qualified a functional assay to assess the potential of ADA to inhibit the neutralizing function of HIV-1 bnAbs. Using a modified version of the validated TZM-bl HIV-1 neutralization assay, murine anti-idiotype antibodies were utilized to optimize and evaluate parameters of linearity, range, limit of detection, specificity, and precision for measuring inhibitory ADA activity against multiple HIV-1 bnAbs that are in clinical development. We further demonstrate the utility of this assay for detecting naturally occurring ADA responses in non-human primates receiving passive administration of human bnAbs. This functional assay format complements binding-antibody ADA strategies being developed for HIV-1 bnAbs, and when utilized together, will support a multi-tiered approach for ADA testing that is compliant with Good Clinical Laboratory Practice (GCLP) procedures and FDA guidance. [ABSTRACT FROM AUTHOR]

HIV- and SIV-envelope (Env) trimers are both extensively glycosylated, and antibodies identified to date have been unable to fully neutralize SIV mac239. Here, we report the isolation, structure, and glycan interactions of antibody ITS90.03, a monoclonal antibody that completely neutralized the highly neutralization-resistant isolate, SIV mac239. The co-crystal structure of a fully glycosylated SIV mac239 -gp120 core in complex with rhesus CD4 and the antigen-binding fragment of ITS90.03 at 2.5-Å resolution revealed that ITS90 recognized an epitope comprised of 45% glycan. SIV-gp120 core, rhesus CD4, and their complex could each be aligned structurally to their human counterparts. The structure revealed that glycans masked most of the SIV Env protein surface, with ITS90 targeting a glycan hole, which is occupied in ∼83% of SIV strains by glycan N 238. Overall, the SIV glycan shield appears to functionally resemble its HIV counterpart in coverage of spike, shielding from antibody, and modulation of receptor accessibility. • Identified ITS90.03, a monoclonal antibody that fully neutralizes SIV mac239 • Crystalized fully glycosylated SIV gp120 with CD4 and ITS90.03 • The ITS90 lineage targets a hole in the SIV mac239 glycan shield • The SIV-glycan shield functionally resembles its HIV counterpart Nonhuman primate antibodies identified to date have been unable to fully neutralize SIV. Gorman et al. present the antibody ITS90.03, isolated from an infected animal, which fully neutralizes SIV mac239. They solved the ITS90.03 structure in complex with a CD4-bound, fully glycosylated SIV mac239 gp120 and found that the SIV-glycan shield functionally resembles its HIV counterpart, with ITS90.03 targeting a glycan hole. [ABSTRACT FROM AUTHOR]