Early Outpatient Treatment for Covid-19 with Convalescent Plasma.

BACKGROUND: Polyclonal convalescent plasma may be obtained from donors who have recovered from coronavirus disease 2019 (Covid-19). The efficacy of this plasma in preventing serious complications in outpatients with recent-onset Covid-19 is uncertain. METHODS: In this multicenter, double-blind, randomized, controlled trial, we evaluated the efficacy and safety of Covid-19 convalescent plasma, as compared with control plasma, in symptomatic adults (≥18 years of age) who had tested positive for severe acute respiratory syndrome coronavirus 2, regardless of their risk factors for disease progression or vaccination status. Participants were enrolled within 8 days after symptom onset and received a transfusion within 1 day after randomization. The primary outcome was Covid-19-related hospitalization within 28 days after transfusion. RESULTS: Participants were enrolled from June 3, 2020, through October 1, 2021. A total of 1225 participants underwent randomization, and 1181 received a transfusion. In the prespecified modified intention-to-treat analysis that included only participants who received a transfusion, the primary outcome occurred in 17 of 592 participants (2.9%) who received convalescent plasma and 37 of 589 participants (6.3%) who received control plasma (absolute risk reduction, 3.4 percentage points; 95% confidence interval, 1.0 to 5.8; P = 0.005), which corresponded to a relative risk reduction of 54%. Evidence of efficacy in vaccinated participants cannot be inferred from these data because 53 of the 54 participants with Covid-19 who were hospitalized were unvaccinated and 1 participant was partially vaccinated. A total of 16 grade 3 or 4 adverse events (7 in the convalescent-plasma group and 9 in the control-plasma group) occurred in participants who were not hospitalized. CONCLUSIONS: In participants with Covid-19, most of whom were unvaccinated, the administration of convalescent plasma within 9 days after the onset of symptoms reduced the risk of disease progression leading to hospitalization. (Funded by the Department of Defense and others; CSSC-004 ClinicalTrials.gov number, NCT04373460.).

Internalization of HIV-1 by Phagocytes Is Increased When Virions Are Opsonized with Multimeric Antibody in the Presence of Complement.

The low abundance of envelope spikes and the inability of IgG to aggregate virions render HIV-1 an inadequate target for antibody-mediated clearance by phagocytes. In an attempt to improve the ability of antibody to mediate the internalization of HIV-1 virions, we generated multimers of the broadly neutralizing HIV-1-specific monoclonal antibody (MAb) VRC01 using site-directed mutagenesis of the Fc segment. We then measured virion internalization using primary human monocytes and neutrophils. We found that, in the absence of complement, immune complexes consisting of HIV-1 virions and VRC01 multimers were slightly more efficiently internalized than were complexes formed with monomeric VRC01. The presence of complement, however, greatly augmented internalization of immune complexes formed with the multimeric MAb but had little impact on monomeric MAb-mediated internalization. Multimerization and the presence of complement overcome the limited ability of monomeric antibody to mediate internalization of HIV-1 virions and may thus provide a therapeutic approach to clearing virus. IMPORTANCE Antibody-mediated internalization of HIV-1 by phagocytes, a potential mechanism for clearing virus, is very inefficient. In an effort to improve viral clearance, we produced a multimeric form of the broadly neutralizing monoclonal antibody VRC01. We found that VRC01 antibody multimers (primarily hexamers) were only slightly more efficient in mediating HIV-1 internalization than was monomeric VRC01. However, the addition of complement resulted in substantially greater internalization of multimer-opsonized virus. In contrast, complement had little if any impact on internalization of monomer-opsonized virus. Therefore, antibody multimerization in combination with complement may overcome the limited ability of monomeric antibody to mediate internalization of HIV-1 virions. Our findings may provide a therapeutic approach to clearing virus.

Vaccination against SARS-CoV-2 using extracellular blebs derived from spike protein-expressing dendritic cells.

COVID-19 has caused significant morbidity and mortality worldwide but also accelerated the clinical use of emerging vaccine formulations. To address the current shortcomings in the prevention and treatment of SARS-CoV-2 infection, this study developed a novel vaccine platform that closely mimics dendritic cells (DCs) in antigen presentation and T-cell stimulation in a cell-free and tunable manner. Genetically engineered DCs that express the SARS-CoV-2 spike protein (S) were chemically converted into extracellular blebs (EBs). The resulting EBs elicited potentially protective humoral immunity in vivo, indicated by the production of antibodies that potently neutralized S-pseudotyped virus, presenting EBs as a promising and safe vaccine.

Transfusion reactions associated with COVID-19 convalescent plasma in outpatient clinical trials.

BACKGROUND: COVID-19 convalescent plasma (CCP) is an important therapeutic option for outpatients at high risk of hospitalization from SARS-CoV-2 infection. We assessed the safety of outpatient CCP transfusions administered during clinical trials. STUDY DESIGN AND METHODS: We analyzed data pertaining to transfusion-related reactions from two randomized controlled trials in the U.S. that evaluated the efficacy of CCP versus control plasma in various ambulatory settings. Multivariable logistic regression was used to assess whether CCP was associated with transfusion reactions, after adjusting for potential confounders. RESULTS: The combined study reported 79/1351 (5.9%) adverse events during the transfusion visit, with the majority 62/1351 (4.6%) characterized by mild, allergic-type findings of urticaria, and/or pruritus consistent with minor allergic transfusion reactions; the other reported events were attributed to the patients' underlying disease, COVID-19, or vasovagal in nature. We found no difference in the likelihood of allergic transfusion reactions between those receiving CCP versus control plasma (adjusted odds ratio [AOR], 0.75; 95% CI, 0.43-1.31). Risk of urticaria and/or pruritus increased with a pre-existing diagnosis of asthma (AOR, 2.33; 95% CI, 1.16-4.67). We did not observe any CCP-attributed antibody disease enhancement in participants with COVID-19 or increased risk of infection. There were no life-threatening severe transfusion reactions and no patients required hospitalization related to transfusion-associated complications. DISCUSSION: Outpatient plasma administration was safely performed for nearly 1400 participants. CCP is a safe therapeutic option for outpatients at risk of hospitalization from COVID-19.

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.

Diverse antiviral IgG effector activities are predicted by unique biophysical antibody features.

BACKGROUND: The critical role of antibody Fc-mediated effector functions in immune defense has been widely reported in various viral infections. These effector functions confer cellular responses through engagement with innate immune cells. The precise mechanism(s) by which immunoglobulin G (IgG) Fc domain and cognate receptors may afford protection are poorly understood, however, in the context of HIV/SHIV infections. Many different in vitro assays have been developed and utilized to measure effector functions, but the extent to which these assays capture distinct antibody activities has not been fully elucidated. RESULTS: In this study, six Fc-mediated effector function assays and two biophysical antibody profiling assays were performed on a common set of samples from HIV-1 infected and vaccinated subjects. Biophysical antibody profiles supported robust prediction of diverse IgG effector functions across distinct Fc-mediated effector function assays. While a number of assays showed correlated activities, supervised machine learning models indicated unique antibody features as primary contributing factors to the associated effector functions. Additional experiments established the mechanistic relevance of relationships discovered using this unbiased approach. CONCLUSIONS: In sum, this study provides better resolution on the diversity and complexity of effector function assays, offering a clearer perspective into this family of antibody mechanisms of action to inform future HIV-1 treatment and vaccination strategies.

Impact of Th1 CD4 Follicular Helper T Cell Skewing on Antibody Responses to an HIV-1 Vaccine in Rhesus Macaques.

Generating durable humoral immunity through vaccination depends upon effective interactions of follicular helper T (Tfh) cells with germinal center (GC) B cells. Th1 polarization of Tfh cells is an important process shaping the success of Tfh-GC B cell interactions by influencing costimulatory and cytokine-dependent Tfh help to B cells. However, the question remains as to whether adjuvant-dependent modulation of Tfh cells enhances HIV-1 vaccine-induced antienvelope (anti-Env) antibody responses. We investigated whether an HIV-1 vaccine platform designed to increase the number of Th1-polarized Tfh cells enhances the magnitude and quality of anti-Env antibodies. Utilizing a novel interferon-induced protein 10 (IP-10)-adjuvanted HIV-1 DNA prime followed by a monophosphoryl lipid A and QS-21 (MPLA+QS-21)-adjuvanted Env protein boost (DIP-10 PALFQ) in macaques, we observed higher anti-Env serum IgG titers with greater cross-clade reactivity, specificity for V1V2, and effector functions than in macaques primed with DNA lacking IP-10 and boosted with MPLA-plus-alum-adjuvanted Env protein (DPALFA) The DIP-10 PALFQ vaccine regimen elicited higher anti-Env IgG1 and lower IgG4 antibody levels in serum, showing for the first time that adjuvants can dramatically impact the IgG subclass profile in macaques. The DIP-10 PALFQ regimen also increased vaginal and rectal IgA antibodies to a greater extent. Within lymph nodes, we observed augmented GC B cell responses and the promotion of Th1 gene expression profiles in GC Tfh cells. The frequency of GC Tfh cells correlated with both the magnitude and avidity of anti-Env serum IgG. Together, these data suggest that adjuvant-induced stimulation of Th1-Tfh cells is an effective strategy for enhancing the magnitude and quality of anti-Env antibody responses.IMPORTANCE The results of the RV144 trial demonstrated that vaccination could prevent HIV transmission in humans and that longevity of anti-Env antibodies may be key to this protection. Efforts to improve upon the prime-boost vaccine regimen used in RV144 have indicated that booster immunizations can increase serum anti-Env antibody titers but only transiently. Poor antibody durability hampers efforts to develop an effective HIV-1 vaccine. This study was designed to identify the specific elements involved in the immunological mechanism necessary to produce robust HIV-1-specific antibodies in rhesus macaques. By clearly defining immune-mediated pathways that improve the magnitude and functionality of the anti-HIV-1 antibody response, we will have the foundation necessary for the rational development of an HIV-1 vaccine.

Expression of CD40L by the ALVAC-Simian Immunodeficiency Virus Vector Abrogates T Cell Responses in Macaques.

Immunization with recombinant ALVAC/gp120 alum vaccine provided modest protection from human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) acquisition in humans and macaques. Vaccine-mediated protection was associated with the elicitation of IgG against the envelope V2 loop and of envelope-specific CD4+ T cell responses. We hypothesized that the simultaneous expression of the costimulatory molecule CD40L (CD154) by the ALVAC-HIV vector could increase both protective humoral and cellular responses. We engineered an ALVAC-SIV coexpressing CD40L with SIVmac251 (ALVAC-SIV/CD40L) gag, pol, and env genes. We compared its immunogenicity in macaques with that of a canonical ALVAC-SIV, with both given as a vector-prime/gp120 in alum boost strategy. The ALVAC-SIV/CD40L was superior to the ALVAC-SIV regimen in inducing binding and tier 1 neutralizing antibodies against the gp120. The increase in humoral responses was associated with the expression of the membrane-bound form of the CD40L by CD4+ T cells in lymph nodes. Unexpectedly, the ALVAC-SIV/CD40L vector had a blunting effect on CD4+ Th1 helper responses and instead favored the induction of myeloid-derived suppressor cells, the immune-suppressive interleukin-10 (IL-10) cytokine, and the down-modulatory tryptophan catabolism. Ultimately, this strategy failed to protect macaques from SIV acquisition. Taken together, these results underlie the importance of balanced vaccine-induced activating versus suppressive immune responses in affording protection from HIV.IMPORTANCE CD40-CD40 ligand (CD40L) interaction is crucial for inducing effective cytotoxic and humoral responses against pathogens. Because of its immunomodulatory function, CD40L has been used to enhance immune responses to vaccines, including candidate vaccines for HIV. The only successful vaccine ever tested in humans utilized a strategy combining canarypox virus-based vector (ALVAC) together with an envelope protein (gp120) adjuvanted in alum. This strategy showed limited efficacy in preventing HIV-1/SIV acquisition in humans and macaques. In both species, protection was associated with vaccine-induced antibodies against the HIV envelope and CD4+ T cell responses, including type 1 antiviral responses. In this study, we tested whether augmenting CD40L expression by coexpressing it with the ALVAC vector could increase the protective immune responses. Although coexpression of CD40L did increase humoral responses, it blunted type 1 CD4+ T cell responses against the SIV envelope protein and failed to protect macaques from viral infection.

Antibody Responses Elicited by Immunization with BG505 Trimer Immune Complexes.

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.IMPORTANCE Immunodominant epitopes may suppress immune responses to more desirable determinants, such as those that elicit potentially protective neutralizing antibody responses. To overcome this problem, we attempted to mask immunodominant glycan holes by immunizing rabbits with ICs consisting of the BG505 SOSIP.664 gp140 trimer and MAbs that targeted the glycan holes. We found that IC vaccination likely diverted antibody responses, to some extent, away from the glycan holes and toward other regions of the trimer. IC vaccination resulted in slower decay of HIV-1-specific antibodies than did immunization with uncomplexed trimer. We did not observe a widening of the breadth or an increase in the potency of neutralizing antibody responses compared to uncomplexed trimers. Our results suggest that selective epitope dampening of BG505 trimers by ICs is rather ineffective. However, IC vaccination may represent a novel means of increasing the duration of vaccine-induced antibody responses.

Replication-Competent NYVAC-KC Yields Improved Immunogenicity to HIV-1 Antigens in Rhesus Macaques Compared to Nonreplicating NYVAC.

As part of the continuing effort to develop an effective HIV vaccine, we generated a poxviral vaccine vector (previously described) designed to improve on the results of the RV144 phase III clinical trial. The construct, NYVAC-KC, is a replication-competent, attenuated recombinant of the vaccinia virus strain NYVAC. NYVAC is a vector that has been used in many previous clinical studies but is replication deficient. Here, we report a side-by-side comparison of replication-restricted NYVAC and replication-competent NYVAC-KC in a nonhuman primate study, which utilized a prime-boost regimen similar to that of RV144. NYVAC-C and NYVAC-C-KC express the HIV-1 antigens gp140, and Gag/Gag-Pol-Nef-derived virus-like particles (VLPs) from clade C and were used as the prime, with recombinant virus plus envelope protein used as the boost. In nearly every T and B cell immune assay against HIV-1, including neutralization and antibody binding, NYVAC-C-KC induced a greater immune response than NYVAC-C, indicating that replication competence in a poxvirus may improve upon the modestly successful regimen used in the RV144 clinical trial.IMPORTANCE Though the RV144 phase III clinical trial showed promise that an effective vaccine against HIV-1 is possible, a successful vaccine will require improvement over the vaccine candidate (ALVAC) used in the RV144 study. With that goal in mind, we have tested in nonhuman primates an attenuated but replication-competent vector, NYVAC-KC, in direct comparison to its parental vector, NYVAC, which is replication restricted in human cells, similar to the ALVAC vector used in RV144. We have utilized a prime-boost regimen for administration of the vaccine candidate that is similar to the one used in the RV144 study. The results of this study indicate that a replication-competent poxvirus vector may improve upon the effectiveness of the RV144 clinical trial vaccine candidate.

Priming with a Potent HIV-1 DNA Vaccine Frames the Quality of Immune Responses prior to a Poxvirus and Protein Boost.

The use of heterologous immunization regimens and improved vector systems has led to increases in immunogenicity of HIV-1 vaccine candidates in nonhuman primates. In order to resolve interrelations between different delivery modalities, three different poxvirus boost regimens were compared. Three groups of rhesus macaques were each primed with the same DNA vaccine encoding Gag, Pol, Nef, and gp140. The groups were then boosted with either the vaccinia virus strain NYVAC or a variant with improved replication competence in human cells, termed NYVAC-KC. The latter was administered either by scarification or intramuscularly. Finally, macaques were boosted with adjuvanted gp120 protein to enhance humoral responses. The regimen elicited very potent CD4+ and CD8+ T cell responses in a well-balanced manner, peaking 2 weeks after the boost. T cells were broadly reactive and polyfunctional. All animals exhibited antigen-specific humoral responses already after the poxvirus boost, which further increased following protein administration. Polyclonal reactivity of IgG antibodies was highest against HIV-1 clade C Env proteins, with considerable cross-reactivity to other clades. Substantial effector functional activities (antibody-dependent cell-mediated cytotoxicity and antibody-dependent cell-mediated virus inhibition) were observed in serum obtained after the last protein boost. Notably, major differences between the groups were absent, indicating that the potent priming induced by the DNA vaccine initially framed the immune responses in such a way that the subsequent boosts with NYVAC and protein led only to an increase in the response magnitudes without skewing the quality. This study highlights the importance of selecting the best combination of vector systems in heterologous prime-boost vaccination regimens.IMPORTANCE The evaluation of HIV vaccine efficacy trials indicates that protection would most likely correlate with a polyfunctional immune response involving several effector functions from all arms of the immune system. Heterologous prime-boost regimens have been shown to elicit vigorous T cell and antibody responses in nonhuman primates that, however, qualitatively and quantitatively differ depending on the respective vector systems used. The present study evaluated a DNA prime and poxvirus and protein boost regimen and compared how two poxvirus vectors with various degrees of replication capacity and two different delivery modalities-conventional intramuscular delivery and percutaneous delivery by scarification-impact several immune effectors. It was found that despite the different poxvirus boosts, the overall immune responses in the three groups were similar, suggesting the potent DNA priming as the major determining factor of immune responses. These findings emphasize the importance of selecting optimal priming agents in heterologous prime-boost vaccination settings.

Combination Adenovirus and Protein Vaccines Prevent Infection or Reduce Viral Burden after Heterologous Clade C Simian-Human Immunodeficiency Virus Mucosal Challenge.

HIV vaccine development is focused on designing immunogens and delivery methods that elicit protective immunity. We evaluated a combination of adenovirus (Ad) vectors expressing HIV 1086.C (clade C) envelope glycoprotein (Env), SIV Gag p55, and human pegivirus GBV-C E2 glycoprotein. We compared replicating simian (SAd7) with nonreplicating human (Ad4) adenovirus-vectored vaccines paired with recombinant proteins in a novel prime-boost regimen in rhesus macaques, with the goal of eliciting protective immunity against SHIV challenge. In both vaccine groups, plasma and buccal Env-specific IgG, tier 1 heterologous neutralizing antibodies, and antibody-dependent cell-mediated viral inhibition were readily generated. High Env-specific T cell responses elicited in all vaccinees were significantly greater than responses targeting Gag. After three intrarectal exposures to heterologous tier 1 clade C SHIV, all 10 sham-vaccinated controls were infected, whereas 4/10 SAd7- and 3/10 Ad4-vaccinated macaques remained uninfected or maintained tightly controlled plasma viremia. Time to infection was significantly delayed in SAd7-vaccinated macaques compared to the controls. Cell-associated and plasma virus levels were significantly lower in each group of vaccinated macaques compared to controls; the lowest plasma viral burden was found in animals vaccinated with the SAd7 vectors, suggesting superior immunity conferred by the replicating simian vectors. Furthermore, higher V1V2-specific binding antibody titers correlated with viral control in the SAd7 vaccine group. Thus, recombinant Ad plus protein vaccines generated humoral and cellular immunity that was effective in either protecting from SHIV acquisition or significantly reducing viremia in animals that became infected, consequently supporting additional development of replicating Ad vectors as HIV vaccines.IMPORTANCE There is a well-acknowledged need for an effective AIDS vaccine that protects against HIV infection and limits in vivo viral replication and associated pathogenesis. Although replicating virus vectors have been advanced as HIV vaccine platforms, there have not been any direct comparisons of the replicating to the nonreplicating format. The present study directly compared the replicating SAd7 to nonreplicating Ad4 vectors in macaques and demonstrated that in the SAd7 vaccine group, the time to infection was significantly delayed compared to the control group, and V1V2 Env-specific binding antibodies correlated with viral outcomes. Viral control was significantly enhanced in vaccinated macaques compared to controls, and in infected SAd7-vaccinated macaques compared to Ad4-vaccinated macaques, suggesting that this vector may have conferred more effective immunity. Because blocking infection is so difficult with current vaccines, development of a vaccine that can limit viremia if infection occurs would be valuable. These data support further development of replicating adenovirus vectors.

Human immunodeficiency virus type-1 (HIV-1) evades antibody-dependent phagocytosis.

Fc gamma receptor (FcyR)-mediated antibody functions play a crucial role in preventing HIV infection. One such function, antibody-dependent phagocytosis (ADP), is thought to be involved in controlling other viral infections, but its role in HIV infection is unknown. We measured the ability of HIV-specific polyclonal and monoclonal antibodies (mAbs) to mediate the internalization of HIV-1 virions and HIV-1-decorated cells by phagocytes. To measure ADP of virions, we primarily used a green-fluorescent protein-expressing molecular clone of HIV-1JRFL, an R5, clinical isolate, in combination with polyclonal HIVIG or mAbs known to capture and/or neutralize HIV-1. THP-1 and U937 cells, as well as freshly isolated primary monocytes from healthy individuals, were used as phagocytic effector cells, and uptake of virions was measured by cytometry. We surprisingly found minimal or no ADP of virions with any of the antibodies. However, after coating virions with gp41 or with gp41-derived peptides, gp41- (but not gp120-) specific mAbs efficiently mediated phagocytosis. We estimated that a minimum of a few hundred gp41 molecules were needed for successful phagocytosis, which is similar to the number of envelope spikes on viruses that are readily phagocytosed (e.g. influenza virus). Furthermore, by employing fluorescence correlation spectroscopy, a well-established technique to measure particle sizes and aggregation phenomena, we found a clear association between virus aggregation and ADP. In contrast to virions themselves, virion-decorated cells were targets for ADP or trogocytosis in the presence of HIV-specific antibodies. Our findings indicate that ADP of virions may not play a role in preventing HIV infection, likely due to the paucity of trimers and the consequent inability of virion-bound antibody to cross-link FcyRs on phagocytes. However, ADP or trogocytosis could play a role in clearing HIV-infected cells and cells on the verge of infection.

Diversity of Antiviral IgG Effector Activities Observed in HIV-Infected and Vaccinated Subjects.

Diverse Ab effector functions mediated by the Fc domain have been commonly associated with reduced risk of infection in a growing number of nonhuman primate and human clinical studies. This study evaluated the anti-HIV Ab effector activities in polyclonal serum samples from HIV-infected donors, VAX004 vaccine recipients, and healthy HIV-negative subjects using a variety of primary and cell line-based assays, including Ab-dependent cellular cytotoxicity (ADCC), Ab-dependent cell-mediated viral inhibition, and Ab-dependent cellular phagocytosis. Additional assay characterization was performed with a panel of Fc-engineered variants of mAb b12. The goal of this study was to characterize different effector functions in the study samples and identify assays that might most comprehensively and dependably capture Fc-mediated Ab functions mediated by different effector cell types and against different viral targets. Deployment of such assays may facilitate assessment of functionally unique humoral responses and contribute to identification of correlates of protection with potential mechanistic significance in future HIV vaccine studies. Multivariate and correlative comparisons identified a set of Ab-dependent cell-mediated viral inhibition and phagocytosis assays that captured different Ab activities and were distinct from a group of ADCC assays that showed a more similar response profile across polyclonal serum samples. The activities of a panel of b12 monoclonal Fc variants further identified distinctions among the ADCC assays. These results reveal the natural diversity of Fc-mediated Ab effector responses among vaccine recipients in the VAX004 trial and in HIV-infected subjects, and they point to the potential importance of polyfunctional Ab responses.

Boosting of ALVAC-SIV Vaccine-Primed Macaques with the CD4-SIVgp120 Fusion Protein Elicits Antibodies to V2 Associated with a Decreased Risk of SIVmac251 Acquisition.

The recombinant ALVAC vaccine coupled with the monomeric gp120/alum protein have decreased the risk of HIV and SIV acquisition. Ab responses to the V1/V2 regions have correlated with a decreased risk of virus acquisition in both humans and macaques. We hypothesized that the breadth and functional profile of Abs induced by an ALVAC/envelope protein regimen could be improved by substituting the monomeric gp120 boost, with the full-length single-chain (FLSC) protein. FLSC is a CD4-gp120 fusion immunogen that exposes cryptic gp120 epitopes to the immune system. We compared the immunogenicity and relative efficiency of an ALVAC-SIV vaccine boosted either with bivalent FLSC proteins or with monomeric gp120 in alum. FLSC was superior to monomeric gp120 in directing Abs to the C3 α2 helix, the V5 loop, and the V3 region that contains the putative CCR5 binding site. In addition, FLSC boosting elicited significantly higher binding Abs to V2 and increased both the Ab-dependent cellular cytotoxicity activity and the breadth of neutralizing Abs. However, the FLSC vaccine regimen demonstrated only a trend in vaccine efficacy, whereas the monomeric gp120 regimen significantly decreased the risk of SIVmac251 acquisition. In both vaccine regimens, anti-V2 Abs correlated with a decreased risk of virus acquisition but differed with regard to systemic or mucosal origin. In the FLSC regimen, serum Abs to V2 correlated, whereas in the monomeric gp120 regimen, V2 Abs in rectal secretions, the site of viral challenge, were associated with efficacy.

Vaccine protection against acquisition of neutralization-resistant SIV challenges in rhesus monkeys.

Preclinical studies of human immunodeficiency virus type 1 (HIV-1) vaccine candidates have typically shown post-infection virological control, but protection against acquisition of infection has previously only been reported against neutralization-sensitive virus challenges. Here we demonstrate vaccine protection against acquisition of fully heterologous, neutralization-resistant simian immunodeficiency virus (SIV) challenges in rhesus monkeys. Adenovirus/poxvirus and adenovirus/adenovirus-vector-based vaccines expressing SIV(SME543) Gag, Pol and Env antigens resulted in an 80% or greater reduction in the per-exposure probability of infection against repetitive, intrarectal SIV(MAC251) challenges in rhesus monkeys. Protection against acquisition of infection showed distinct immunological correlates compared with post-infection virological control and required the inclusion of Env in the vaccine regimen. These data demonstrate the proof-of-concept that optimized HIV-1 vaccine candidates can block acquisition of stringent, heterologous, neutralization-resistant virus challenges in rhesus monkeys.

Glycan modulation and sulfoengineering of anti-HIV-1 monoclonal antibody PG9 in plants.

Broadly neutralizing anti-HIV-1 monoclonal antibodies, such as PG9, and its derivative RSH hold great promise in AIDS therapy and prevention. An important feature related to the exceptional efficacy of PG9 and RSH is the presence of sulfated tyrosine residues in their antigen-binding regions. To maximize antibody functionalities, we have now produced glycan-optimized, fucose-free versions of PG9 and RSH in Nicotiana benthamiana. Both antibodies were efficiently sulfated in planta on coexpression of an engineered human tyrosylprotein sulfotransferase, resulting in antigen-binding and virus neutralization activities equivalent to PG9 synthesized by mammalian cells ((CHO)PG9). Based on the controlled production of both sulfated and nonsulfated variants in plants, we could unequivocally prove that tyrosine sulfation is critical for the potency of PG9 and RSH. Moreover, the fucose-free antibodies generated in N. benthamiana are capable of inducing antibody-dependent cellular cytotoxicity, an activity not observed for (CHO)PG9. Thus, tailoring of the antigen-binding site combined with glycan modulation and sulfoengineering yielded plant-produced anti-HIV-1 antibodies with effector functions superior to PG9 made in CHO cells.

Relationship between Vaccine-Induced Antibody Capture of Infectious Virus and Infection Outcomes following Repeated Low-Dose Rectal Challenges with Simian Immunodeficiency Virus SIVmac251.

UNLABELLED: Antibodies are known to enhance in vitro infection by human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV). We measured the ability of antibodies induced by ALVAC-SIV/gp120 vaccination, given with alum or MF59 adjuvant, to capture infectious SIVmac251 and determined the association between capture and infection outcomes following low-dose, repeated rectal challenge of rhesus macaques. We found that capture correlated with the number of transmitted/founder (T/F) variants that established infection, such that animals whose plasma captured more virus were infected with a higher number of T/F strains. Capture also correlated with results of Env binding assays, indicating that greater immunogenicity resulted in greater capture. Although vaccination elicited negligible neutralizing activity against the challenge strain (50% inhibitory dilutions of >1/80 in all cases), animals with low capture and whose plasma, at a fixed dilution, inhibited a higher fraction of virus were infected at a lower rate than animals with high capture and low neutralization (P = 0.039); only animals with the low capture/high neutralization response profile were protected compared with unvaccinated control animals (P = 0.026). In a sieve analysis, high capture and low capture were distinguishable on the basis of polymorphisms in the V1 loop of Env at amino acids 144 and 145. Our results indicate that vaccine-induced antibody that binds to and captures infectious virus but does not inhibit its infectivity may enhance the likelihood of infection following rectal challenge with SIVmac251. Higher immunogenicity resulting in better antibody capture but similar anti-infectivity may not improve vaccine efficacy. IMPORTANCE: Vaccines generally prevent viral infections by eliciting antibodies that inhibit virus infectivity. However, antibodies, including those induced by vaccination, have the potential to enhance, rather than prevent infection. We measured the ability of vaccine-induced antibodies to capture infectious simian immunodeficiency virus (SIV) and explored the relationship between virus capture and infection outcomes. We found that capture correlated with the number of SIV variants that established infection, such that animals whose plasma captured more virus were infected with a higher number of unique strains. In addition, animals whose sera had high capture but weak anti-infectivity activity were infected at a higher rate than were animals with low capture and stronger anti-infectivity activity. These results suggest that vaccines that induce antibodies that bind to and capture infectious virus but do not inhibit virus infectivity will not be effective in preventing infection.

Potential To Streamline Heterologous DNA Prime and NYVAC/Protein Boost HIV Vaccine Regimens in Rhesus Macaques by Employing Improved Antigens.

UNLABELLED: In a follow-up to the modest efficacy observed in the RV144 trial, researchers in the HIV vaccine field seek to substantiate and extend the results by evaluating other poxvirus vectors and combinations with DNA and protein vaccines. Earlier clinical trials (EuroVacc trials 01 to 03) evaluated the immunogenicity of HIV-1 clade C GagPolNef and gp120 antigens delivered via the poxviral vector NYVAC. These showed that a vaccination regimen including DNA-C priming prior to a NYVAC-C boost considerably enhanced vaccine-elicited immune responses compared to those with NYVAC-C alone. Moreover, responses were improved by using three as opposed to two DNA-C primes. In the present study, we assessed in nonhuman primates whether such vaccination regimens can be streamlined further by using fewer and accelerated immunizations and employing a novel generation of improved DNA-C and NYVAC-C vaccine candidates designed for higher expression levels and more balanced immune responses. Three different DNA-C prime/NYVAC-C+ protein boost vaccination regimens were tested in rhesus macaques. All regimens elicited vigorous and well-balanced CD8(+)and CD4(+)T cell responses that were broad and polyfunctional. Very high IgG binding titers, substantial antibody-dependent cellular cytotoxicity (ADCC), and modest antibody-dependent cell-mediated virus inhibition (ADCVI), but very low neutralization activity, were measured after the final immunizations. Overall, immune responses elicited in all three groups were very similar and of greater magnitude, breadth, and quality than those of earlier EuroVacc vaccines. In conclusion, these findings indicate that vaccination schemes can be simplified by using improved antigens and regimens. This may offer a more practical and affordable means to elicit potentially protective immune responses upon vaccination, especially in resource-constrained settings. IMPORTANCE: Within the EuroVacc clinical trials, we previously assessed the immunogenicity of HIV clade C antigens delivered in a DNA prime/NYVAC boost regimen. The trials showed that the DNA prime crucially improved the responses, and three DNA primes with a NYVAC boost appeared to be optimal. Nevertheless, T cell responses were primarily directed toward Env, and humoral responses were modest. The aim of this study was to assess improved antigens for the capacity to elicit more potent and balanced responses in rhesus macaques, even with various simpler immunization regimens. Our results showed that the novel antigens in fact elicited larger numbers of T cells with a polyfunctional profile and a good Env-GagPolNef balance, as well as high-titer and Fc-functional antibody responses. Finally, comparison of the different schedules indicates that a simpler regimen of only two DNA primes and one NYVAC boost in combination with protein may be very efficient, thus showing that the novel antigens allow for easier immunization protocols.