The elicitation of broadly neutralizing antibodies (bNAbs) against the HIV-1 envelope glycoprotein (Env) trimer remains a major vaccine challenge. Most cross-conserved protein determinants are ...occluded by self-N-glycan shielding, limiting B cell recognition of the underlying polypeptide surface. The exceptions to the contiguous glycan shield include the conserved receptor CD4 binding site (CD4bs) and glycoprotein (gp)41 elements proximal to the furin cleavage site. Accordingly, we performed heterologous trimer-liposome prime:boosting in rabbits to drive B cells specific for cross-conserved sites. To preferentially expose the CD4bs to B cells, we eliminated proximal N-glycans while maintaining the native-like state of the cleavage-independent NFL trimers, followed by gradual N-glycan restoration coupled with heterologous boosting. This approach successfully elicited CD4bs-directed, cross-neutralizing Abs, including one targeting a unique glycan-protein epitope and a bNAb (87% breadth) directed to the gp120:gp41 interface, both resolved by high-resolution cryoelectron microscopy. This study provides proof-of-principle immunogenicity toward eliciting bNAbs by vaccination.
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•Removal of N-glycans proximal to the CD4 binding site increases B cell accessibility•Heterologous Env trimer-liposome regimen drives B cells to cross-conserved sites•Vaccine elicitation of an N-glycan-dependent CD4 binding site neutralizing antibody•Elicitation of an interface-directed antibody with 87% HIV neutralization breadth
Eliciting broadly neutralizing HIV antibodies by vaccination remains a challenge. Dubrovskaya et al. use an immunization regimen incorporating targeted N-glycan removal and heterologous prime:boosting with NFL trimer-liposomes in rabbits to elicit broadly neutralizing responses to cross-conserved HIV-1 epitopes, including an antibody with 87% neutralization breadth. Further structural analyses highlight similarities between the vaccine-elicited antibodies and the human broadly neutralizing antibodies.
The potency and breadth of the recently isolated neutralizing human monoclonal antibodies to HIV-1 have stimulated interest in their use to prevent or to treat HIV-1 infection. Due to the ...antigenically diverse nature of the HIV-1 envelope (Env), no single antibody is highly active against all viral strains. While the physical combination of two broadly neutralizing antibodies (bNAbs) can improve coverage against the majority of viruses, the clinical-grade manufacturing and testing of two independent antibody products are time and resource intensive. In this study, we constructed bispecific immunoglobulins (IgGs) composed of independent antigen-binding fragments with a common Fc region. We developed four different bispecific IgG variants that included antibodies targeting four major sites of HIV-1 neutralization. We show that these bispecific IgGs display features of both antibody specificities and, in some cases, display improved coverage over the individual parental antibodies. All four bispecific IgGs neutralized 94% to 97% of antigenically diverse viruses in a panel of 206 HIV-1 strains. Among the bispecific IgGs tested, VRC07 × PG9-16 displayed the most favorable neutralization profile. It was superior in breadth to either of the individual antibodies, neutralizing 97% of viruses with a median 50% inhibitory concentration (IC50) of 0.055 μg/ml. This bispecific IgG also demonstrated in vivo pharmacokinetic parameters comparable to those of the parental bNAbs when administered to rhesus macaques. These results suggest that IgG-based bispecific antibodies are promising candidates for the prevention and treatment of HIV-1 infection in humans.
To prevent or treat HIV-1 infection, antibodies must potently neutralize nearly all strains of HIV-1. Thus, the physical combination of two or more antibodies may be needed to broaden neutralization coverage and diminish the possibility of viral resistance. A bispecific antibody that has two different antibody binding arms could potentially display neutralization characteristics better than those of any single parental antibody. Here we show that bispecific antibodies contain the binding specificities of the two parental antibodies and that a single bispecific antibody can neutralize 97% of viral strains with a high overall potency. These findings support the use of bispecific antibodies for the prevention or treatment of HIV-1 infection.
Summary
VRC‐HIVMAB060‐00‐AB (VRC01) is a broadly neutralizing HIV‐1 monoclonal antibody (mAb) isolated from the B cells of an HIV‐infected patient. It is directed against the HIV‐1 CD4 binding site ...and is capable of potently neutralizing the majority of diverse HIV‐1 strains. This Phase I dose‐escalation study in healthy adults was conducted at the National Institutes of Health (NIH) Clinical Center (Bethesda, MD, USA). Primary objectives were the safety, tolerability and pharmacokinetics (PK) of VRC01 intravenous (i.v.) infusion at 5, 20 or 40 mg/kg, given either once (20 mg/kg) or twice 28 days apart (all doses), and of subcutaneous (s.c.) delivery at 5 mg/kg compared to s.c. placebo given twice, 28 days apart. Cumulatively, 28 subjects received 43 VRC01 and nine received placebo administrations. There were no serious adverse events or dose‐limiting toxicities. Mean 28‐day serum trough concentrations after the first infusion were 35 and 57 μg/ml for groups infused with 20 mg/kg (n = 8) and 40 mg/kg (n = 5) doses, respectively. Mean 28‐day trough concentrations after the second infusion were 56 and 89 μg/ml for the same two doses. Over the 5–40 mg/kg i.v. dose range (n = 18), the clearance was 0·016 l/h and terminal half‐life was 15 days. After infusion VRC01 retained expected neutralizing activity in serum, and anti‐VRC01 antibody responses were not detected. The human monoclonal antibody (mAb) VRC01 was well tolerated when delivered i.v. or s.c. The mAb demonstrated expected half‐life and pharmacokinetics for a human immunoglobulin G. The safety and PK results support and inform VRC01 dosing schedules for planning HIV‐1 prevention efficacy studies.
The vaccine-mediated elicitation of antibodies (Abs) capable of neutralizing diverse HIV-1 strains has been a long-standing goal. To understand how broadly neutralizing antibodies (bNAbs) can be ...elicited, we identified, characterized, and tracked five neutralizing Ab lineages targeting the HIV-1-fusion peptide (FP) in vaccinated macaques over time. Genetic and structural analyses revealed two of these lineages to belong to a reproducible class capable of neutralizing up to 59% of 208 diverse viral strains. B cell analysis indicated each of the five lineages to have been initiated and expanded by FP-carrier priming, with envelope (Env)-trimer boosts inducing cross-reactive neutralization. These Abs had binding-energy hotspots focused on FP, whereas several FP-directed Abs induced by immunization with Env trimer-only were less FP-focused and less broadly neutralizing. Priming with a conserved subregion, such as FP, can thus induce Abs with binding-energy hotspots coincident with the target subregion and capable of broad neutralization.
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•Vaccine elicitation of FP-directed antibody that neutralizes up to 59% of HIV•Development for five NHP lineages of vaccine-induced broadly neutralizing antibodies•FP-carrier effective at priming and Env-trimer effective at maturing•Priming with a target subregion induces antibodies with target-interaction hotspots
A cross-clade, cross-reactive HIV-1 neutralizing antibody with ∼59% neutralization breadth is elicited in macaques using a fusion-peptide-primed vaccine regimen, which focuses antibody-binding energy on a conserved viral epitope. Further phylogenetic antibody analysis provides insight into the eclipse phase of B cell development.
Background. The development of an effective human immunodeficiency virus (HIV) vaccine is a high global priority. Here, we report the safety, tolerability, and immunogenicity of a ...replication‐defective recombinant adenovirus serotype 5 (rAd5) vector HIV‐1 candidate vaccine. Methods. The vaccine is a mixture of 4 rAd5 vectors that express HIV‐1 subtype B Gag‐Pol fusion protein and envelope (Env) from subtypes A, B, and C. Healthy, uninfected adults were randomized to receive 1 intramuscular injection of placebo (n=6) or vaccine at dose levels of 109 (n=10), 1010 (n=10), or 1011 (n=10) particle units and were followed for 24 weeks to assess immunogenicity and safety. Results. The vaccine was well tolerated but was associated with more reactogenicity at the highest dose. At week 4, vaccine antigen–specific T cell responses were detected in 28 (93.3%) and 18 (60%) of 30 vaccine recipients for CD4+ and CD8+ T cells, respectively, by intracellular cytokine staining assay and in 22 (73%) of 30 vaccine recipients by enzyme‐linked immunospot assay. Env‐specific antibody responses were detected in 15 (50%) of 30 vaccine recipients by enzyme‐linked immunosorbant assay and in 28 (93.3%) of 30 vaccine recipients by immunoprecipitation followed by Western blotting. No neutralizing antibody was detected. Conclusions. A single injection induced HIV‐1 antigen–specific CD4+ T cell, CD8+ T cell, and antibody responses in the majority of vaccine recipients. This multiclade rAd5 HIV‐1 vaccine is now being evaluated in combination with a multiclade HIV‐1 DNA plasmid vaccine.
Background. Gene‐based vaccine delivery is an important strategy in the development of a preventive vaccine for acquired immunodeficiency syndrome (AIDS). Vaccine Research Center (VRC) 004 is the ...first phase 1 dose‐escalation study of a multiclade HIV‐1 DNA vaccine. Methods. VRC‐HIVDNA009‐00‐VP is a 4‐plasmid mixture encoding subtype B Gag‐Pol‐Nef fusion protein and modified envelope (Env) constructs from subtypes A, B, and C. Fifty healthy, uninfected adults were randomized to receive either placebo (n=10) or study vaccine at 2 mg (n=5), 4 mg (n=20), or 8 mg (n=15) by needle‐free intramuscular injection. Humoral responses (measured by enzyme‐linked immunosorbant assay, Western blotting, and neutralization assay) and T cell responses (measured by enzyme‐linked immunospot assay and intracellular cytokine staining after stimulation with antigen‐specific peptide pools) were measured. Results. The vaccine was well tolerated and induced cellular and humoral responses. The maximal CD4+ and CD8+ T cell responses occurred after 3 injections and were in response to Env peptide pools. The pattern of cytokine expression by vaccine‐induced HIV‐specific T cells evolved over time, with a diminished frequency of interferon‐γ–producing T cells and an increased frequency of interleukin‐2–producing T cells at 1 year. Conclusions. DNA vaccination induced antibody to and T cell responses against 3 major HIV‐1 subtypes and will be further evaluated as a potential component of a preventive AIDS vaccine regimen.
Background. Ebolavirus and Marburgvirus cause severe hemorrhagic fever with high mortality and are potential bioterrorism agents. There are no available vaccines or therapeutic agents. Previous ...clinical trials evaluated transmembrane-deleted and point-mutation Ebolavirus glycoproteins (GPs) in candidate vaccines. Constructs evaluated in this trial encode wild-type (WT) GP from Ebolavirus Zaire and Sudan species and the Marburgvirus Angola strain expressed in a DNA vaccine. Methods. The VRC 206 study evaluated the safety and immunogenicity of these DNA vaccines (4 mg administered intramuscularly by Biojector) at weeks 0, 4, and 8, with a homologous boost at or after week 32. Safety evaluations included solicited reactogenicity and coagulation parameters. Primary immune assessment was done by means of GP-specific enzyme-linked immunosorbent assay. Results. The vaccines were well tolerated, with no serious adverse events; 80% of subjects had positive enzymelinked immunosorbent assay results (≥30) at week 12. The fourth DNA vaccination boosted the immune responses. Conclusions. The investigational Ebolavirus and Marburgvirus WT GP DNA vaccines were safe, well tolerated, and immunogenic in this phase I study. These results will further inform filovirus vaccine research toward a goal of inducing protective immunity by using WT GP antigens in candidate vaccine regimens.
IL-12 is a pivotal cytokine that links the innate and adaptive immune responses. TNF-alpha also plays a key role in orchestrating inflammation and immunity. The reciprocal influence of these two ...inflammatory mediators on each other may have significant impact on the cytokine balance that shapes the type and extent of immune responses. To investigate the relationship between TNF-alpha and IL-12 production, we analyzed the effects of exposure of human monocyte-derived macrophages to TNF-alpha on LPS- or Staphylococcus aureus-induced IL-12 production in the presence or absence of IFN-gamma. TNF-alpha is a potent inhibitor of IL-12 p40 and p70 secretion from human macrophages induced by LPS or S. aureus. IL-10 is not responsible for the TNF-alpha-mediated inhibition of IL-12. TNF-alpha selectively inhibits IL-12 p40 steady-state mRNA, but not those of IL-12 p35, IL-1alpha, IL-1beta, or IL-6. Nuclear run-on analysis identified this specific inhibitory effect at the transcriptional level for IL-12 p40 without down-regulation of the IL-12 p35 gene. The major transcriptional factors identified to be involved in the regulation of IL-12 p40 gene expression by LPS and IFN-gamma, i.e., c-Rel, NF-kappaB p50 and p65, IFN regulatory factor-1, and ets-2, were not affected by TNF-alpha when examined by nuclear translocation and DNA binding. These data demonstrate a selective negative regulation on IL-12 by TNF-alpha, identifying a direct negative feedback mechanism for inflammation-induced suppression of IL-12 gene expression.
The nucleocapsid (N) protein is a structural component of severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) and can induce antibody responses in SARS patients during infection. However, ...it is not known whether SARS-CoV N protein can induce a long persistence of memory T-cell response in human. In this study, we found that peripheral blood mononuclear cells (PBMCs) from fully recovered SARS individuals rapidly produced IFN-γ and IL-2 following stimulation with a pool of overlapping peptides that cover the entire N protein sequence. The N-specific IFN-γ
+CD4
+ T cells were mainly composed of CD45RA
−CCR7
+CD62L
− cells, whereas IFN-γ
+CD8
+ memory T cells were mostly contained within CD45RA
+CCR7
−CD62L
− cell population. Epitope mapping study indicated that a cluster of overlapping peptides located in the C-terminal region (amino acids aa 331 to 362) of N protein contained at least two different T-cell epitopes. The results indicated that human memory T-cell responses specific for SARS-CoV N protein could persist for 2 years in the absence of antigen, which would be a valuable for the design of effective vaccines against SARS-CoV and for basic studies of human T-cell memory.
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