A Group M Consensus Envelope Glycoprotein Induces Antibodies That Neutralize Subsets of Subtype B and C HIV-1 Primary Viruses

HIV-1 subtype C is the most common HIV-1 group M subtype in Africa and many parts of Asia. However, to date HIV-1 vaccine candidate immunogens have not induced potent and broadly neutralizing antibodies against subtype C primary isolates. We have used a centralized gene strategy to address HIV-1 diversity, and generated a group M consensus envelope gene with shortened consensus variable loops (CON-S) for comparative studies with wildtype (WT) Env immunogens. Our results indicate that the consensus HIV-1 group M CON-S Env elicited cross-subtype neutralizing antibodies of similar or greater breadth and titer than the WT Envs tested, indicating the utility of a centralized gene strategy. Our study also shows the feasibility of iterative improvements in Env immunogenicity by rational design of centralized genes

Safety, pharmacokinetics and target engagement of novel RIPK1 inhibitor SAR443060 (DNL747) for neurodegenerative disorders:Randomized, placebo-controlled, double-blind phase I/Ib studies in healthy subjects and patients

RIPK1 is a master regulator of inflammatory signaling and cell death and increased RIPK1 activity is observed in human diseases, including Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS). RIPK1 inhibition has been shown to protect against cell death in a range of preclinical cellular and animal models of diseases. SAR443060 (previously DNL747) is a selective, orally bioavailable, central nervous system (CNS)-penetrant, small-molecule, reversible inhibitor of RIPK1. In three early-stage clinical trials in healthy subjects and patients with AD or ALS (NCT03757325 and NCT03757351), SAR443060 distributed into the cerebrospinal fluid (CSF) after oral administration and demonstrated robust peripheral target engagement as measured by a reduction in phosphorylation of RIPK1 at serine 166 (pRIPK1) in human peripheral blood mononuclear cells compared to baseline. RIPK1 inhibition was generally safe and well-tolerated in healthy volunteers and patients with AD or ALS. Taken together, the distribution into the CSF after oral administration, the peripheral proof-of-mechanism, and the safety profile of RIPK1 inhibition to date, suggest that therapeutic modulation of RIPK1 in the CNS is possible, conferring potential therapeutic promise for AD and ALS, as well as other neurodegenerative conditions. However, SAR443060 development was discontinued due to long-term nonclinical toxicology findings, although these nonclinical toxicology signals were not observed in the short duration dosing in any of the three early-stage clinical trials. The dose-limiting toxicities observed for SAR443060 preclinically have not been reported for other RIPK1-inhibitors, suggesting that these toxicities are compound-specific (related to SAR443060) rather than RIPK1 pathway-specific

Cross-reactive monoclonal antibodies to multiple HIV-1 subtype and SIVcpz envelope glycoproteins

The extraordinarily high level of genetic variation of HIV-1 env genes poses a challenge to obtain antibodies that cross-react with multiple subtype Env glycoproteins. To determine if cross-reactive monoclonal antibodies (mAbs) to highly conserved epitopes in HIV-1 envelope glycoproteins can be induced, we immunized mice with wild-type or consensus HIV-1 Env proteins and characterized a panel of ten mAbs that reacted with varying breadth to subtypes A, B, C, D, F, G, CRF01_AE and a highly divergent SIVcpzUS Env proteins by ELISA and Western blot analysis. Two mAbs (3B3 and 16H3) cross–reacted with all tested Env proteins, including SIVcpzUS Env. Surface plasmon resonance analyses showed both 3B3 and 16H3 bound Env proteins with high affinity. However, neither neutralized primary HIV-1 pseudoviruses. These data indicate that broadly-reactive non-neutralizing monoclonal antibodies can be elicited, but that the conserved epitopes that they recognize are not present on functional virion trimers. Nonetheless, such mAbs represent valuable reagents to study the biochemistry and structural biology of Env protein oligomers

Anti-phospholipid human monoclonal antibodies inhibit CCR5-tropic HIV-1 and induce β-chemokines

Traditional antibody-mediated neutralization of HIV-1 infection is thought to result from the binding of antibodies to virions, thus preventing virus entry. However, antibodies that broadly neutralize HIV-1 are rare and are not induced by current vaccines. We report that four human anti-phospholipid monoclonal antibodies (mAbs) (PGN632, P1, IS4, and CL1) inhibit HIV-1 CCR5-tropic (R5) primary isolate infection of peripheral blood mononuclear cells (PBMCs) with 80% inhibitory concentrations of <0.02 to ∼10 µg/ml. Anti-phospholipid mAbs inhibited PBMC HIV-1 infection in vitro by mechanisms involving binding to monocytes and triggering the release of MIP-1α and MIP-1β. The release of these β-chemokines explains both the specificity for R5 HIV-1 and the activity of these mAbs in PBMC cultures containing both primary lymphocytes and monocytes

Envelope Deglycosylation Enhances Antigenicity of HIV-1 gp41 Epitopes for Both Broad Neutralizing Antibodies and Their Unmutated Ancestor Antibodies

The HIV-1 gp41 envelope (Env) membrane proximal external region (MPER) is an important vaccine target that in rare subjects can elicit neutralizing antibodies. One mechanism proposed for rarity of MPER neutralizing antibody generation is lack of reverted unmutated ancestor (putative naive B cell receptor) antibody reactivity with HIV-1 envelope. We have studied the effect of partial deglycosylation under non-denaturing (native) conditions on gp140 Env antigenicity for MPER neutralizing antibodies and their reverted unmutated ancestor antibodies. We found that native deglycosylation of clade B JRFL gp140 as well as group M consensus gp140 Env CON-S selectively increased the reactivity of Env with the broad neutralizing human mAbs, 2F5 and 4E10. Whereas fully glycosylated gp140 Env either did not bind (JRFL), or weakly bound (CON-S), 2F5 and 4E10 reverted unmutated ancestors, natively deglycosylated JRFL and CON-S gp140 Envs did bind well to these putative mimics of naive B cell receptors. These data predict that partially deglycoslated Env would bind better than fully glycosylated Env to gp41-specific naïve B cells with improved immunogenicity. In this regard, immunization of rhesus macaques demonstrated enhanced immunogenicity of the 2F5 MPER epitope on deglyosylated JRFL gp140 compared to glycosylated JRFL gp140. Thus, the lack of 2F5 and 4E10 reverted unmutated ancestor binding to gp140 Env may not always be due to lack of unmutated ancestor antibody reactivity with gp41 peptide epitopes, but rather, may be due to glycan interference of binding of unmutated ancestor antibodies of broad neutralizing mAb to Env gp41

Induction of Antibodies in Rhesus Macaques That Recognize a Fusion-Intermediate Conformation of HIV-1 gp41

A component to the problem of inducing broad neutralizing HIV-1 gp41 membrane proximal external region (MPER) antibodies is the need to focus the antibody response to the transiently exposed MPER pre-hairpin intermediate neutralization epitope. Here we describe a HIV-1 envelope (Env) gp140 oligomer prime followed by MPER peptide-liposomes boost strategy for eliciting serum antibody responses in rhesus macaques that bind to a gp41 fusion intermediate protein. This Env-liposome immunization strategy induced antibodies to the 2F5 neutralizing epitope 664DKW residues, and these antibodies preferentially bound to a gp41 fusion intermediate construct as well as to MPER scaffolds stabilized in the 2F5-bound conformation. However, no serum lipid binding activity was observed nor was serum neutralizing activity for HIV-1 pseudoviruses present. Nonetheless, the Env-liposome prime-boost immunization strategy induced antibodies that recognized a gp41 fusion intermediate protein and was successful in focusing the antibody response to the desired epitope

Functional, Non-Clonal IgMa-Restricted B Cell Receptor Interactions with the HIV-1 Envelope gp41 Membrane Proximal External Region

The membrane proximal external region (MPER) of HIV-1 gp41 has several features that make it an attractive antibody-based vaccine target, but eliciting an effective gp41 MPER-specific protective antibody response remains elusive. One fundamental issue is whether the failure to make gp41 MPER-specific broadly neutralizing antibodies like 2F5 and 4E10 is due to structural constraints with the gp41 MPER, or alternatively, if gp41 MPER epitope-specific B cells are lost to immunological tolerance. An equally important question is how B cells interact with, and respond to, the gp41 MPER epitope, including whether they engage this epitope in a non-canonical manner i.e., by non-paratopic recognition via B cell receptors (BCR). To begin understanding how B cells engage the gp41 MPER, we characterized B cell-gp41 MPER interactions in BALB/c and C57BL/6 mice. Surprisingly, we found that a significant (∼7%) fraction of splenic B cells from BALB/c, but not C57BL/6 mice, bound the gp41 MPER via their BCRs. This strain-specific binding was concentrated in IgMhi subsets, including marginal zone and peritoneal B1 B cells, and correlated with enriched fractions (∼15%) of gp41 MPER-specific IgM secreted by in vitro-activated splenic B cells. Analysis of Igha (BALB/c) and Ighb (C57BL/6) congenic mice demonstrated that gp41 MPER binding was controlled by determinants of the Igha locus. Mapping of MPER gp41 interactions with IgMa identified MPER residues distinct from those to which mAb 2F5 binds and demonstrated the requirement of Fc CH regions. Importantly, gp41 MPER ligation produced detectable BCR-proximal signaling events, suggesting that interactions between gp41 MPER and IgMa determinants may elicit partial B cell activation. These data suggest that low avidity, non-paratopic interactions between the gp41 MPER and membrane Ig on naïve B cells may interfere with or divert bnAb responses

Structural constraints of vaccine-induced tier-2 autologous HIV neutralizing antibodies targeting the receptor-binding site.

CAPRISA, 2017.Abstract available in pdf

Amino acid changes in the HIV-1 gp41 membrane proximal region control virus neutralization sensitivity.

CAPRISA, 2016.Abstract available in pdf

Zika virus protection by a single low dose nucleoside modified mRNA vaccination

Zika virus (ZIKV) has recently emerged as an explosive pandemic associated with severe neuropathology in newborns and adults1. There are no ZIKV-specific treatments or preventatives; thus, development of a safe and effective vaccine is a high priority. Messenger RNA (mRNA) has emerged as a versatile and highly effective platform to deliver vaccine antigens and therapeutic proteins2,3. Here, we demonstrate that a single low-dose intradermal immunization with lipid nanoparticle-encapsulated nucleoside-modified mRNA (mRNA-LNP) encoding the pre-membrane and envelope (prM-E) glycoproteins of a 2013 ZIKV outbreak strain elicited potent and durable neutralizing antibody responses in mice and non-human primates. Immunization with 30 μg of nucleoside-modified ZIKV mRNA-LNPs protected mice from ZIKV challenges at 2 weeks or 5 months post-vaccination, and a single dose of 50 μg was sufficient to protect non-human primates from a challenge at 5 weeks post-vaccination. These data demonstrate that nucleoside-modified mRNA-LNPs elicit rapid and durable protective immunity and thus represent a new and promising vaccine candidate for the global fight against ZIKV