Complement contributes to antibody-mediated protection against repeated SHIV challenge.

The first clinical efficacy trials of a broadly neutralizing antibody (bNAb) resulted in less benefit than expected and suggested that improvements are needed to prevent HIV infection. While considerable effort has focused on optimizing neutralization breadth and potency, it remains unclear whether augmenting the effector functions elicited by broadly neutralizing antibodies (bNAbs) may also improve their clinical potential. Among these effector functions, complement-mediated activities, which can culminate in the lysis of virions or infected cells, have been the least well studied. Here, functionally modified variants of the second-generation bNAb 10-1074 with ablated and enhanced complement activation profiles were used to examine the role of complement-associated effector functions. When administered prophylactically against simian-HIV challenge in rhesus macaques, more bNAb was required to prevent plasma viremia when complement activity was eliminated. Conversely, less bNAb was required to protect animals from plasma viremia when complement activity was enhanced. These results suggest that complement-mediated effector functions contribute to in vivo antiviral activity, and that their engineering may contribute to the further improvements in the efficacy of antibody-mediated prevention strategies.

Non-neutralizing antibodies targeting the immunogenic regions of HIV-1 envelope reduce mucosal infection and virus burden in humanized mice.

Antibodies are principal immune components elicited by vaccines to induce protection from microbial pathogens. In the Thai RV144 HIV-1 vaccine trial, vaccine efficacy was 31% and the sole primary correlate of reduced risk was shown to be vigorous antibody response targeting the V1V2 region of HIV-1 envelope. Antibodies against V3 also were inversely correlated with infection risk in subsets of vaccinees. Antibodies recognizing these regions, however, do not exhibit potent neutralizing activity. Therefore, we examined the antiviral potential of poorly neutralizing monoclonal antibodies (mAbs) against immunodominant V1V2 and V3 sites by passive administration of human mAbs to humanized mice engrafted with CD34+ hematopoietic stem cells, followed by mucosal challenge with an HIV-1 infectious molecular clone expressing the envelope of a tier 2 resistant HIV-1 strain. Treatment with anti-V1V2 mAb 2158 or anti-V3 mAb 2219 did not prevent infection, but V3 mAb 2219 displayed a superior potency compared to V1V2 mAb 2158 in reducing virus burden. While these mAbs had no or weak neutralizing activity and elicited undetectable levels of antibody-dependent cellular cytotoxicity (ADCC), V3 mAb 2219 displayed a greater capacity to bind virus- and cell-associated HIV-1 envelope and to mediate antibody-dependent cellular phagocytosis (ADCP) and C1q complement binding as compared to V1V2 mAb 2158. Mutations in the Fc region of 2219 diminished these effector activities in vitro and lessened virus control in humanized mice. These results demonstrate the importance of Fc functions other than ADCC for antibodies without potent neutralizing activity.

Polyfunctional Tier 2-Neutralizing Antibodies Cloned following HIV-1 Env Macaque Immunization Mirror Native Antibodies in a Human Donor.

Vaccine efforts to combat HIV are challenged by the global diversity of viral strains and shielding of neutralization epitopes on the viral envelope glycoprotein trimer. Even so, the isolation of broadly neutralizing Abs from infected individuals suggests the potential for eliciting protective Abs through vaccination. This study reports a panel of 58 mAbs cloned from a rhesus macaque (Macaca mulatta) immunized with envelope glycoprotein immunogens curated from an HIV-1 clade C-infected volunteer. Twenty mAbs showed neutralizing activity, and the strongest neutralizer displayed 92% breadth with a median IC50 of 1.35 µg/ml against a 13-virus panel. Neutralizing mAbs predominantly targeted linear epitopes in the V3 region in the cradle orientation (V3C) with others targeting the V3 ladle orientation (V3L), the CD4 binding site (CD4bs), C1, C4, or gp41. Nonneutralizing mAbs bound C1, C5, or undetermined conformational epitopes. Neutralization potency strongly correlated with the magnitude of binding to infected primary macaque splenocytes and to the level of Ab-dependent cellular cytotoxicity, but did not predict the degree of Ab-dependent cellular phagocytosis. Using an individualized germline gene database, mAbs were traced to 23 of 72 functional IgHV alleles. Neutralizing V3C Abs displayed minimal nucleotide somatic hypermutation in the H chain V region (3.77%), indicating that relatively little affinity maturation was needed to achieve in-clade neutralization breadth. Overall, this study underscores the polyfunctional nature of vaccine-elicited tier 2-neutralizing V3 Abs and demonstrates partial reproduction of the human donor's humoral immune response through nonhuman primate vaccination.

Virus Control in Vaccinated Rhesus Macaques Is Associated with Neutralizing and Capturing Antibodies against the SHIV Challenge Virus but Not with V1V2 Vaccine-Induced Anti-V2 Antibodies Alone.

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 vaccine-elicited 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.

Antibodies Tip the Balance Towards an HIV Cure.

Long-term drug therapy for HIV-1 infection can prevent both disease progression and virus transmission, but treatment does not eradicate the virus. A new gene therapy study (Immunity 2019;50:567-575.e5) using human antibodies shows significant promise for a functional cure in a non-human primate model.

An HIV Vaccine Targeting the V2 Region of the HIV Envelope Induces a Highly Durable Polyfunctional Fc-Mediated Antibody Response in Rhesus Macaques.

The HIV vaccine field now recognizes the potential importance of generating polyfunctional antibodies (Abs). The only clinical HIV vaccine trial to date to show significant efficacy (RV144) found that reduced infection rates correlated with the level of nonneutralizing Abs specific for the V2 region of the envelope glycoprotein. We have conducted a comprehensive preclinical reverse vaccinology-based vaccine program that has included the design and production and testing of numerous scaffolded V2 region immunogens. The most immunogenic vaccine regimen in nonhuman primates among those studied as part of this program consisted of a cocktail of three immunogens presenting V2 from different viruses and clades in the context of different scaffolds. Presently we demonstrate that the V2-specific Ab response from this regimen was highly durable and functionally diverse for the duration of the study (25 weeks after the final immunization). The total IgG binding response at this late time point exhibited only an ∼5× reduction in potency. Three immunizations appeared essential for the elicitation of a strong Ab-dependent cellular cytotoxicity (ADCC) response for all animals, as opposed to the Ab-dependent cellular phagocytosis (ADCP) and virus capture responses, which were comparably potent after only 2 immunizations. All functionalities measured were highly durable through the study period. Therefore, testing this vaccine candidate for its protective capacity is warranted.IMPORTANCE The only HIV vaccine trial for which protective efficacy was detected correlated this efficacy with V2-specific Abs that were effectively nonneutralizing. This result has fueled a decade of HIV vaccine research focused on designing an HIV vaccine capable of eliciting V2-focused, polyfunctional Abs that effectively bind HIV and trigger various leukocytes to kill the virus and restrict viral spread. From the numerous vaccine candidates designed and tested as part of our V2-focused preclinical vaccine program, we have identified immunogens and a vaccine regimen that induces a highly durable and polyfunctional V2-focused Ab response in rhesus macaques, described herein.

Use of broadly neutralizing antibodies for HIV-1 prevention.

Antibodies have a long history in antiviral therapy, but until recently, they have not been actively pursued for HIV-1 due to modest potency and breadth of early human monoclonal antibodies (MAbs) and perceived insurmountable technical, financial, and logistical hurdles. Recent advances in the identification and characterization of MAbs with the ability to potently neutralize diverse HIV-1 isolates have reinvigorated discussion and testing of these products in humans, since new broadly neutralizing MAbs (bnMAbs) are more likely to be effective against worldwide strains of HIV-1. In animal models, there is abundant evidence that bnMAbs can block infection in a dose-dependent manner, and the more potent bnMAbs will allow clinical testing at infusion doses that are practically achievable. Moreover, recent advances in antibody engineering are providing further improvements in MAb potency, breadth, and half-life. This review summarizes the current state of the field of bnMAb protection in animal models as well as a review of variables that are critical for antiviral activity. Several bnMAbs are currently in clinical testing, and we offer perspectives on their use as pre-exposure prophylaxis (PrEP), potential benefits beyond sterilizing immunity, and a discussion of future approaches to engineer novel molecules.

Divergent HIV-1-Directed Immune Responses Generated by Systemic and Mucosal Immunization with Replicating Single-Cycle Adenoviruses in Rhesus Macaques.

Most human immunodeficiency virus type 1 (HIV-1) infections begin at mucosal surfaces. Providing a barrier of protection at these may assist in combating the earliest events in infection. Systemic immunization by intramuscular (i.m.) injection can drive mucosal immune responses, but there are data suggesting that mucosal immunization can better educate these mucosal immune responses. To test this, rhesus macaques were immunized with replicating single-cycle adenovirus (SC-Ad) vaccines expressing clade B HIV-1 gp160 by the intranasal (i.n.) and i.m. routes to compare mucosal and systemic routes of vaccination. SC-Ad vaccines generated significant circulating antibody titers against Env after a single i.m. immunization. Switching the route of second immunization with the same SC-Ad serotype allowed a significant boost in these antibody levels. When these animals were boosted with envelope protein, envelope-binding antibodies were amplified 100-fold, but qualitatively different immune responses were generated. Animals immunized by only the i.m. route had high peripheral T follicular helper (pTfh) cell counts in blood but low Tfh cell counts in lymph nodes. Conversely, animals immunized by the i.n. route had high Tfh cell counts in lymph nodes but low pTfh cell counts in the blood. Animals immunized by only the i.m. route had lower antibody-dependent cellular cytotoxicity (ADCC) antibody activity, whereas animals immunized by the mucosal i.n. route had higher ADCC antibody activity. When these Env-immunized animals were challenged rectally with simian-human immunodeficiency virus (SHIV) strain SF162P3 (SHIVSF162P3), they all became infected. However, mucosally SC-Ad-immunized animals had lower viral loads in their gastrointestinal tracts. These data suggest that there may be benefits in educating the immune system at mucosal sites during HIV vaccination.IMPORTANCE HIV-1 infections usually start at a mucosal surface after sexual contact. Creating a barrier of protection at these mucosal sites may be a good strategy for to protect against HIV-1 infections. While HIV-1 enters at mucosa, most vaccines are not delivered here. Most are instead injected into the muscle, a site well distant and functionally different than mucosal tissues. This study tested if delivering HIV vaccines at mucosa or in the muscle makes a difference in the quality, quantity, and location of immune responses against the virus. These data suggest that there are indeed advantages to educating the immune system at mucosal sites with an HIV-1 vaccine.

Cloning and functional testing of rhesus macaque (Macaca mulatta) IL-9 and IL-33.

BACKGROUND: IL-9 and IL-33 can profoundly influence immune responses. As a necessary first step toward defining their impact in the rhesus macaque model, we confirmed their endogenous expression and sequence identity and generated expression vectors for the recombinant expression of rhesus IL-9 and IL-33. METHODS: RT-PCR and Sanger sequencing was used to define the expression and sequences for rhesus IL-9 and IL-33. The resulting recombinant cytokines were tested by ELISA and proliferation assays. RESULTS: Full-length rhesus IL-9 and the mature form of rhesus IL-33 share 78% and 73% nucleotide similarity, respectively, with humans. Both cytokines are expressed in lymphocytes, with IL-9 expression also evident in CD4+ T cells. Recombinantly expressed rhesus IL-9 and IL-33 were each biologically active in vitro, including enhancing the proliferation of a rhesus B cell line. CONCLUSIONS: The recombinant rhesus IL-9 and IL-33 constructs produce biologically active cytokines that can act upon rhesus B cells.

Reduced Cell-Associated DNA and Improved Viral Control in Macaques following Passive Transfer of a Single Anti-V2 Monoclonal Antibody and Repeated Simian/Human Immunodeficiency Virus Challenges.

A high level of V1V2-specific IgG antibodies (Abs) in vaccinees' sera was the only independent variable that correlated with a reduced risk of human immunodeficiency virus (HIV) acquisition in the RV144 clinical trial. In contrast, IgG avidity, antibody neutralization, and antibody-dependent cellular cytotoxicity each failed as independent correlates of infection. Extended analyses of RV144 samples demonstrated the antiviral activities of V1V2-specific vaccine-induced antibodies. V2-specific antibodies have also been associated with protection from simian immunodeficiency virus (SIV), and the V2i-specific subset of human monoclonal antibodies (MAbs), while poor neutralizers, mediates Fc-dependent antiviral functions in vitro The objective of this study was to determine the protective efficacy of a V2i-specific human MAb, 830A, against mucosal simian/human immunodeficiency virus (SHIV) challenge. V2i MAb binding sites overlap the integrin binding site in the V2 region and are similar to the epitopes bound by antibodies associated with reduced HIV infection rates in RV144. Because the IgG3 subclass was a correlate of reduced infection rates in RV144, we compared passive protection by both IgG1 and IgG3 subclasses of V2i MAb 830A. This experiment represents the first in vivo test of the hypothesis emanating from RV144 and SIV studies that V2i Abs can reduce the risk of infection. The results show that passive transfer with a single V2i MAb, IgG1 830A, reduced plasma and peripheral blood mononuclear cell (PBMC) virus levels and decreased viral DNA in lymphoid tissues compared to controls, but too few animals remained uninfected to achieve significance in reducing the risk of infection. Based on these findings, we conclude that V2i antibodies can impede virus seeding following mucosal challenge, resulting in improved virus control.IMPORTANCE Since the results of the HIV RV144 clinical trial were reported, there has been significant interest in understanding how protection was mediated. Antibodies directed to a subregion of the envelope protein called V1V2 were directly correlated with a reduced risk, and surprisingly low virus neutralization was observed. To determine whether these antibodies alone could mediate protection, we used a human monoclonal antibody directed to V2 with properties similar to those elicited in the vaccine trial for passive infusions in rhesus macaques and challenge with SHIV. The single V2 antibody at the dose given did not significantly reduce the number of infections, but there was a significant reduction in the seeding of virus to the lymph nodes and a decrease in plasma viremia in the HIV antibody-infused macaques compared with the control antibody-infused animals. This finding shows that V2 antibodies mediate antiviral activities in vivo that could contribute to a protective HIV vaccine.

Achieving Potent Autologous Neutralizing Antibody Responses against Tier 2 HIV-1 Viruses by Strategic Selection of Envelope Immunogens.

Advancement in immunogen selection and vaccine design that will rapidly elicit a protective Ab response is considered critical for HIV vaccine protective efficacy. Vaccine-elicited Ab responses must therefore have the capacity to prevent infection by neutralization-resistant phenotypes of transmitted/founder (T/F) viruses that establish infection in humans. Most vaccine candidates to date have been ineffective at generating Abs that neutralize T/F or early variants. In this study, we report that coimmunizing rhesus macaques with HIV-1 gp160 DNA and gp140 trimeric protein selected from native envelope gene sequences (envs) induced neutralizing Abs against Tier 2 autologous viruses expressing cognate envelope (Env). The Env immunogens were selected from envs emerging during the earliest stages of neutralization breadth developing within the first 2 years of infection in two clade B-infected human subjects. Moreover, the IgG responses in macaques emulated the targeting to specific regions of Env known to be associated with autologous and heterologous neutralizing Abs developed within the human subjects. Furthermore, we measured increasing affinity of macaque polyclonal IgG responses over the course of the immunization regimen that correlated with Tier 1 neutralization. In addition, we report firm correlations between Tier 2 autologous neutralization and Tier 1 heterologous neutralization, as well as overall TZM-bl breadth scores. Additionally, the activation of Env-specific follicular helper CD4 T cells in lymphocytes isolated from inguinal lymph nodes of vaccinated macaques correlated with Tier 2 autologous neutralization. These results demonstrate the potential for native Env derived from subjects at the time of neutralization broadening as effective HIV vaccine elements.

The MHC class II cofactor HLA-DM interacts with Ig in B cells.

B cells internalize extracellular Ag into endosomes using the Ig component of the BCR. In endosomes, Ag-derived peptides are loaded onto MHC class II proteins. How these pathways intersect remains unclear. We find that HLA-DM (DM), a catalyst for MHC class II peptide loading, coprecipitates with Ig in lysates from human tonsillar B cells and B cell lines. The molecules in the Ig/DM complexes have mature glycans, and the complexes colocalize with endosomal markers in intact cells. A larger fraction of Ig precipitates with DM after BCR crosslinking, implying that complexes can form when DM meets endocytosed Ig. In vitro, in the endosomal pH range, soluble DM directly binds the Ig Fab domain and increases levels of free Ag released from immune complexes. Taken together, these results argue that DM and Ig intersect in the endocytic pathway of B cells with potential functional consequences.

Envelope variants circulating as initial neutralization breadth developed in two HIV-infected subjects stimulate multiclade neutralizing antibodies in rabbits.

UNLABELLED: Identifying characteristics of the human immunodeficiency virus type 1 (HIV-1) envelope that are effective in generating broad, protective antibodies remains a hurdle to HIV vaccine design. Emerging evidence of the development of broad and potent neutralizing antibodies in HIV-infected subjects suggests that founder and subsequent progeny viruses may express unique antigenic motifs that contribute to this developmental pathway. We hypothesize that over the course of natural infection, B cells are programmed to develop broad antibodies by exposure to select populations of emerging envelope quasispecies variants. To test this hypothesis, we identified two unrelated subjects whose antibodies demonstrated increasing neutralization breadth against a panel of HIV-1 isolates over time. Full-length functional env genes were cloned longitudinally from these subjects from months after infection through 2.6 to 5.8 years of infection. Motifs associated with the development of breadth in published, cross-sectional studies were found in both subjects. We compared the immunogenicity of envelope vaccines derived from time points obtained during and after broadening of neutralization activity within these subjects. Rabbits were coimmunized four times with selected multiple gp160 DNAs and gp140-trimeric envelope proteins. The affinity of the polyclonal response increased as a function of boosting. The most rapid and persistent neutralization of multiclade tier 1 viruses was elicited by envelopes that were circulating in plasma at time points prior to the development of 50% neutralization breadth in both human subjects. The breadth elicited in rabbits was not improved by exposure to later envelope variants. These data have implications for vaccine development in describing a target time point to identify optimal envelope immunogens. IMPORTANCE: Vaccine protection against viral infections correlates with the presence of neutralizing antibodies; thus, vaccine components capable of generating potent neutralization are likely to be critical constituents in an effective HIV vaccine. However, vaccines tested thus far have elicited only weak antibody responses and very modest, waning protection. We hypothesized that B cells develop broad antibodies by exposure to the evolving viral envelope population and tested this concept using multiple envelopes from two subjects who developed neutralization breadth within a few years of infection. We compared different combinations of envelopes from each subject to identify the most effective immunogens and regimens. In each subject, use of HIV envelopes circulating during the early development and maturation of breadth generated more-potent antibodies that were modestly cross neutralizing. These data suggest a new approach to identifying envelope immunogens that may be more effective in generating protective antibodies in humans.

Emergence of broadly neutralizing antibodies and viral coevolution in two subjects during the early stages of infection with human immunodeficiency virus type 1.

UNLABELLED: Delineating the key early events that lead to the development of broadly neutralizing anti-HIV-1 antibodies during natural infection may help guide the development of immunogens and vaccine regimens to prevent HIV-1 infection. In this study, we monitored two HIV-1-positive subjects, VC20013 and VC10014, over the course of infection from before they developed broadly neutralizing antibody (bNAb) activity until several years after neutralizing breadth was detected in plasma. Both subjects developed bNAb activity after approximately 1 year postinfection, which ultimately mapped to the membrane-proximal external region (MPER) in VC20013 and an epitope that overlaps the CD4 receptor binding site in VC10014. In subject VC20013, we were able to identify anti-MPER activity in the earliest plasma sample that exhibited no bNAb activity, indicating that this epitope specificity was acquired very early on, but that it was initially not able to mediate neutralization. Escape mutations within the bNAb epitopes did not arise in the circulating envelopes until bNAb activity was detectable in plasma, indicating that this early response was not sufficient to drive viral escape. As bNAb activity began to emerge in both subjects, we observed a simultaneous increase in autologous antienvelope antibody binding affinity, indicating that antibody maturation was occurring as breadth was developing. Our findings illustrate one potential mechanism by which bNAbs develop during natural infection in which an epitope target is acquired very early on during the course of infection but require time and maturation to develop into broadly neutralizing activity. IMPORTANCE: One major goal of HIV-1 vaccine research is the development of a vaccine that can elicit broadly neutralizing antibodies (bNAbs). Although no such vaccine exists, bNAbs develop in approximately 20% of HIV-1-infected subjects, providing a prototype of the bNAbs that must be reelicited by vaccine. Thus, there is significant interest in understanding the mechanisms by which bNAbs develop during the course of infection. We studied the timing, epitope specificity, and evolution of the bNAb responses in two HIV-1-positive patients who developed bNAb activity within the first several years after infection. In one subject, antibodies to a broadly neutralizing epitope developed very early but were nonneutralizing. After several months, neutralizing activity developed, and the virus mutated to escape their activity. Our study highlights one mechanism for the development of bNAbs where early epitope acquisition followed by sufficient time for antibody maturation drives the epitope-specific antibody response toward broadly neutralizing activity.

Simplifying the synthesis of SIgA: combination of dIgA and rhSC using affinity chromatography.

The mucosal epithelia together with adaptive immune responses, such as local production and secretion of dimeric and polymeric immunoglobulin A (IgA), are a crucial part of the first line of defense against invading pathogens. IgA is primarily secreted as SIgA and plays multiple roles in mucosal defense. The study of SIgA-mediated protection is an important area of research in mucosal immunity but an easy, fast and reproducible method to generate pathogen-specific SIgA in vitro has not been available. We report here a new method to produce SIgA by co-purification of dimeric IgA, containing J chain, and recombinant human SC expressed in CHO cells. We previously reported the generation, production and characterization of the human recombinant monoclonal antibody IgA2 b12. This antibody, derived from the variable regions of the neutralizing anti-HIV-1 mAb IgG1 b12, blocked viral attachment and uptake by epithelial cells in vitro. We used a cloned CHO cell line that expresses monomeric, dimeric and polymeric species of IgA2 b12 for large-scale production of dIgA2 b12. Subsequently, we generated a CHO cell line to express recombinant human secretory component (rhSC). Here, we combined dIgA2 b12 and CHO-expressed rhSC via column chromatography to produce SIgA2 b12 that remains fully intact upon elution with 0.1M citric acid, pH 3.0. We have performed biochemical analysis of the synthesized SIgA to confirm the species is of the expected size and retains the functional properties previously described for IgA2 b12. We show that SIgA2 b12 binds to the HIV-1 gp120 glycoprotein with similar apparent affinity to that of monomeric and dimeric forms of IgA2 b12 and neutralizes HIV-1 isolates with similar potency. An average yield of 6 mg of SIgA2 b12 was achieved from the combination of 20mg of purified dIgA2 b12 and 2L of rhSC-containing CHO cell supernatant. We conclude that synthesized production of stable SIgA can be generated by co-purification. This process introduces a simplified means of generating a variety of pathogen-specific SIgA antibodies for research and clinical applications.

Neutralizing polyclonal IgG present during acute infection prevents rapid disease onset in simian-human immunodeficiency virus SHIVSF162P3-infected infant rhesus macaques.

Simian-human immunodeficiency virus (SHIV) models for human immunodeficiency virus (HIV) infection have been widely used in passive studies with HIV neutralizing antibodies (NAbs) to test for protection against infection. However, because SHIV-infected adult macaques often rapidly control plasma viremia and any resulting pathogenesis is minor, the model has been unsuitable for studying the impact of antibodies on pathogenesis in infected animals. We found that SHIVSF162P3 infection in 1-month-old rhesus macaques not only results in high persistent plasma viremia but also leads to very rapid disease progression within 12 to 16 weeks. In this model, passive transfer of high doses of neutralizing IgG (SHIVIG) prevents infection. Here, we show that at lower doses, SHIVIG reduces both plasma and peripheral blood mononuclear cell (PBMC)-associated viremia and mitigates pathogenesis in infected animals. Moreover, production of endogenous NAbs correlated with lower set-point viremia and 100% survival of infected animals. New SHIV models are needed to investigate whether passively transferred antibodies or antibodies elicited by vaccination that fall short of providing sterilizing immunity impact disease progression or influence immune responses. The 1-month-old rhesus macaque SHIV model of infection provides a new tool to investigate the effects of antibodies on viral replication and clearance, mechanisms of B cell maintenance, and the induction of adaptive immunity in disease progression.

Structure of the Ebola virus glycoprotein bound to an antibody from a human survivor.

Ebola virus (EBOV) entry requires the surface glycoprotein (GP) to initiate attachment and fusion of viral and host membranes. Here we report the crystal structure of EBOV GP in its trimeric, pre-fusion conformation (GP1+GP2) bound to a neutralizing antibody, KZ52, derived from a human survivor of the 1995 Kikwit outbreak. Three GP1 viral attachment subunits assemble to form a chalice, cradled by the GP2 fusion subunits, while a novel glycan cap and projected mucin-like domain restrict access to the conserved receptor-binding site sequestered in the chalice bowl. The glycocalyx surrounding GP is likely central to immune evasion and may explain why survivors have insignificant neutralizing antibody titres. KZ52 recognizes a protein epitope at the chalice base where it clamps several regions of the pre-fusion GP2 to the amino terminus of GP1. This structure provides a template for unravelling the mechanism of EBOV GP-mediated fusion and for future immunotherapeutic development.

Limited or no protection by weakly or nonneutralizing antibodies against vaginal SHIV challenge of macaques compared with a strongly neutralizing antibody.

To guide vaccine design, we assessed whether human monoclonal antibodies (MAbs) b12 and b6 against the CD4 binding site (CD4bs) on HIV-1 gp120 and F240 against an immundominant epitope on gp41 could prevent vaginal transmission of simian HIV (SHIV)-162P4 to macaques. The two anti-gp120 MAbs have similar monomeric gp120-binding properties, measured in vitro, but b12 is strongly neutralizing and b6 is not. F240 is nonneutralizing. Applied vaginally at a high dose, the strongly neutralizing MAb b12 provided sterilizing immunity in seven of seven animals, b6 in zero of five animals, and F240 in two of five animals. Compared with control animals, the protection by b12 achieved statistical significance, whereas that caused by F240 did not. For two of three unprotected F240-treated animals there was a trend toward lowered viremia. The potential protective effect of F240 may relate to the relatively strong ability of this antibody to capture infectious virions. Additional passive transfer experiments also indicated that the ability of the administered anti-gp120 MAbs to neutralize the challenge virus was a critical influence on protection. Furthermore, when data from all of the experiments were combined, there was a significant increase in the number of founder viruses establishing infection in animals receiving MAb b6, compared with other nonprotected macaques. Thus, a gp120-binding, weakly neutralizing MAb to the CD4bs was, at best, completely ineffective at protection. A nonneutralizing antibody to gp41 may have a limited capacity to protect, but the results suggest that the central focus of HIV-1 vaccine research should be on the induction of potently neutralizing antibodies.

Design, synthesis, and biological evaluation of a biyouyanagin compound library.

Modern drug discovery efforts rely, to a large extent, on lead compounds from two classes of small organic molecules; namely, natural products (i.e., secondary metabolites) and designed compounds (i.e., synthetic molecules). In this article, we demonstrate how these two domains of lead compounds can be merged through total synthesis and molecular design of analogs patterned after the targeted natural products, whose promising biological properties provide the motivation. Specifically, the present study targeted the naturally occurring biyouyanagins A and B and their analogs through modular chemical synthesis and led to the discovery of small organic molecules possessing anti-HIV and anti-arenavirus properties.

Sex- and species-associated differences in complement-mediated immunity in humans and rhesus macaques.

The complement system can be viewed as a "moderator" of innate immunity, "instructor" of humoral immunity, and "regulator" of adaptive immunity. While sex is known to affect humoral and cellular immune systems, its impact on complement in humans and rhesus macaques, a commonly used non-human primate model system, has not been well studied. To address this knowledge gap, we analyzed serum samples from 90 humans and 72 rhesus macaques for the abundance and activity of the complement system components. While sequences of cascade proteins were highly conserved, dramatically different levels were observed between species. Whereas the low levels detected in rhesus samples raised questions about the suitability of the test for use with macaque samples, differences in levels of complement proteins were observed in male and female humans. Levels of total and antibody-dependent deposition of C1q and C3b on a glycosylated antigen differed between humans and rhesus, suggesting differential recognition of glycans and balance between classical and alternative activation pathways. Functional differences in complement-mediated lysis of antibody-sensitized cells were observed in multiple assays and showed that human females frequently exhibited higher lytic activity than human males or rhesus macaques, which typically did not exhibit such sex-associated differences. Other differences between species and sexes were observed in more narrow contexts-for only certain antibodies, antigens, or assays. Collectively, these results expand knowledge of sex-associated differences in the complement system in humans, identifying differences absent from rhesus macaques.IMPORTANCEThe complement system is a critical part of host defense to many bacterial, fungal, and viral infections. In parallel, rich epidemiological, clinical, and biomedical research evidence demonstrates that sex is an important biological variable in immunity, and many sex-specific differences in immune system are intimately tied with disease outcomes. This study focuses on the intersection of these two factors to define the impact of sex on complement pathway components and activities. This work expands our knowledge of sex-associated differences in the complement system in humans and also identifies the differences that appear to be absent in rhesus macaques, a popular non-human primate model. Whereas differences between species suggest potential limitations in the ability of macaque model to recapitulate human biology, knowledge of sex-based differences in humans has the potential to inform clinical research and practice.