Faculty Bibliography

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic since December 2019, and with it, a push for innovations in rapid testing and neutralizing antibody treatments in an effort to solve the spread and fatality of the disease. One such solution to both of these prevailing issues is targeting the interaction of SARS-CoV-2 spike receptor binding domain (RBD) with the human angiotensin-converting enzyme 2 (ACE2) receptor protein. Structural studies have shown that the N-terminal alpha-helix comprised of the first 23 residues of ACE2 plays an important role in this interaction. Where it is typical to design a binding domain to fit a target, we have engineered a protein that relies on multivalency rather than the sensitivity of a monomeric ligand to provide avidity to its target by fusing the N-terminal helix of ACE2 to the coiled-coil domain of the cartilage oligomeric matrix protein. The resulting ACE-MAP is able to bind to the SARS-CoV-2 RBD with improved binding affinity, is expressible in E. coli, and is thermally stable and relatively small (62 kDa). These properties suggest ACE-MAP and the MAP scaffold to be a promising route towards developing future diagnostics and therapeutics to SARS-CoV-2.

BACKGROUND:Eleven tau immunoglobulin G (IgG) antibodies have entered clinical trials to treat tauopathies, including Alzheimer's disease, but it is unclear which IgG subclass/subtype has the ideal efficacy and safety profile. Only two subtypes, with or without effector function, have been examined in the clinic and not for the same tau antibody. The few preclinical studies on this topic have only compared two subtypes of one antibody each and have yielded conflicting results. METHODS:subclasses containing identical tau binding domains but differing Fc region. Unmodified sdAbs and their IgG subclasses were tested for efficacy in primary cultures and in vivo microdialysis using JNPL3 tauopathy mice. FINDINGS/RESULTS:subclasses varied greatly within and between sdAbs. For one of them, all its subtypes were non-toxic, only those with effector function cleared tau, and were more effective in vivo than unmodified sdAb. For the other sdAb, all its subtypes were toxic in tauopathy cultures but not in wild-type cells, suggesting that bivalent binding of its tau epitope stabilizes a toxic conformation of tau, with major implications for tau pathogenesis. Likewise, its subclasses were less effective than the unmodified sdAb in clearing tau in vivo. INTERPRETATION/CONCLUSIONS:These findings indicate that tau antibodies with effector function are safe and better at clearing pathological tau than effectorless antibodies, Furthermore, tau antibodies can provide a valuable insight into tau pathogenesis, and some may aggravate it. FUNDING/BACKGROUND:Funding for these studies was provided by the National Institute of Health (R01 AG032611, R01 NS077239, RF1 NS120488, R21 AG 069475, R21 AG 058282, T32AG052909), and the NYU Alzheimer's Disease Center Pilot Grant Program (via P30 AG008051).

The HIV-1 envelope glycoprotein spike is the target of antibodies, and therefore represents the main viral antigen for antibody-based vaccine design. One of the challenges in HIV-1 vaccine development is finding efficient ways for the immune system to recognize and respond to HIV-1 without establishing an infection. Since HIV-1 enters the body at mucosal surfaces, induction of immune response at these sites is a preferred preventive approach. Nasal administration is a very effective route for mucosal immunization since it can stimulate mucosal immune responses both locally and distantly. In this paper, Luna develops a safe, short carbon nanotube (CNT)-based, needle-free delivery platform known as "CNTVac". The size of short CNT was controlled to possess HIV-1 particle-like morphology (100-200 nm) capable of efficiently delivering a broad range of antigens intranasally. PEG-Lipid served as the antigen conformation protector and mucosal barrier penetration enhancer (Schematic Figure) was localized between V1V2 antigens, which caused highly enhanced local IgA and systemic antibody IgG responses in mice and rabbits. The short CNT incorporated with PEG-Lipid could not only serve as efficient delivery system but also reduce the amount of lipid usage in order to balance the vaccine dosage in order to eliminate the potential adverse effect. These data suggest a promising platform technology for vaccine delivery.

V2p and V2i antibodies (Abs) that are specific for epitopes in the V1V2 region of the HIV gp120 envelope (Env) do not effectively neutralize HIV but mediate Fc-dependent anti-viral activities that have been correlated with protection from, or control of HIV, SIV and SHIV infections. Here, we describe a novel molecular toolbox that allows the discrimination of antigenically and functionally distinct polyclonal V2 Ab responses. We identify different patterns of V2 Ab induction by SHIV infection and three separate vaccine regimens that aid in fine-tuning an optimized immunization protocol for inducing V2p and V2i Abs. We observe no, or weak and sporadic V2p and V2i Abs in non-vaccinated SHIV-infected NHPs, but strong V2p and/or V2i Ab responses after immunization with a V2-targeting vaccine protocol. The V2-focused vaccination is superior to both natural infection and to immunization with whole Env constructs for inducing functional V2p- and V2i-specific responses. Strikingly, levels of V2-directed Abs correlate inversely with Abs specific for peptides of V3 and C5. These data demonstrate that a V1V2-targeting vaccine has advantages over the imprecise targeting of SIV/SHIV infections and of whole Env-based immunization regimens for inducing a more focused functional V2p- and V2i-specific Ab response.

Developing a safe and effective malaria vaccine is critical to reducing the spread and resurgence of this deadly disease, especially in children. In recent years, vaccine technology has seen expanded development of subunit protein, peptide, and nucleic acid vaccines. This is due to their inherent safety, the ability to tailor their immune response, simple storage requirements, easier production, and lower expense compared to using attenuated and inactivated organism-based approaches. However, these new vaccine technologies generally have low efficacy. Subunit vaccines, due to their weak immunogenicity, often necessitate advanced delivery vectors and/or the use of adjuvants. A new area of vaccine development involves design of synthetic micro- and nano-particles and adjuvants that can stimulate immune cells directly through their physical and chemical properties. Further, the unique and complex life cycle of the Plasmodium organism, with multiple stages and varying epitopes/antigens presented by the parasite, is another challenge for malaria vaccine development. Targeting multistage antigens simultaneously is therefore critical for an effective malaria vaccine. Here, we rationally design a layer-by-layer (LbL) antigen delivery platform (we called LbL NP) specifically engineered for malaria vaccines. A biocompatible modified chitosan nanoparticle (trimethyl chitosan, TMC) was synthesized and utilized for LbL loading and release of multiple malaria antigens from pre-erythrocytic and erythrocytic stages. LbL NP served as antigen/protein delivery vehicles and were demonstrated to induce the highest Plasmodium falciparum Circumsporozoite Protein (PfCSP) specific T-cell responses in mice studies as compared to multiple controls. From immunogenicity studies, it was concluded that two doses of intramuscular injection with a longer interval (4 weeks) than traditional malaria vaccine candidate dosing would be the vaccination potential for LbL NP vaccine candidates. Furthermore, in PfCSP/Py parasite challenge studies we demonstrated protective efficacy using LbL NP. These LbL NP provided a significant adjuvant effect since they may induce innate immune response that led to a potent adaptive immunity to mediate non-specific anti-malarial effect. Most importantly, the delivery of CSP full-length protein stimulated long-lasting protective immune responses even after the booster immunization 4 weeks later in mice.

We studied the contribution of the light chain to functions of human monoclonal antibodies (mAbs) by measuring the relationships between the rate of mutations and cross-reactivity, binding affinity and neutralization activity. We analyzed 12 mAbs of two clonal families specific to the V2 region of HIV-1 derived from two chronically HIV-1 infected individuals. The clonal mAbs exhibited a range of reactivities, and the clones with superior properties were associated with the rate of mutations and the presence of particular mutated residues in the light chains, but not in the heavy chains. Our observations suggest that for some antibodies, the light chains play a vital role in antibody evolution toward more efficient ones and also suggest the importance of optimal residues rather than the rate of mutations in the variable fragment of the antibody.

Identification of vulnerable sites defined by broadly neutralizing antibodies (bNAbs) on HIV-1 envelope (Env) is crucial for vaccine design, and we present here a vulnerable site defined by bNAb M4008_N1, which neutralizes about 40% of a tier-2 virus panel. A 3.2 Å resolution cryo-EM structure of M4008_N1 in complex with BG505 DS-SOSIP reveals a large, shallow protein epitope surface centered at the V3 crown of gp120 and surrounded by key glycans. M4008_N1 interacts with gp120 primarily through its hammerhead CDR H3 to form a β-sheet interaction with the V3 crown hairpin. This makes M4008_N1 compatible with the closed conformation of the prefusion Env trimer, and thus distinct from other known V3 crown mAbs. This mode of bNAb approaching the immunogenic V3 crown in the native Env trimer suggests a strategy for immunogen design targeting this site of vulnerability.

The repertoire of antibodies (Abs) produced upon vaccination against a particular antigenic site is rarely studied due to the complexity of the immunogens. We received such an opportunity when one rhesus macaque was immunized six times at 0, 4, 10, 16, 32, and 143 weeks with C4-447 peptide containing the 8-mer epitope for human monoclonal Ab (mAb) 447-52D specific to the V3 region of gp120 HIV-1. Strong anti-V3 antibody responses reached 50% binding titer in serum of 10^-5 at week 10 that declined to 10^-3 by week 70. After an additional boost of C4-447 peptide at week 143, titers rebounded to 10^-5 at week 146, or 2.7 years after the first immunization. Using the blood sample at week 146, we produced 41 V3-specific recombinant mAbs by single B cell isolation and cloning. Sequence analysis revealed 21B cell lineages, single and clonally related, based on immunoglobulin gene usage and CDR3s. The broad repertoire of Abs directed to a small antigenic site shows the targeting potency of a vaccine-elicited immune response in rhesus macaques.

Combinational antiretroviral therapy (cART) is the most effective tool to prevent and control HIV-1 infection without an effective vaccine. However, HIV-1 drug resistance mutations (DRMs) and naturally occurring polymorphisms (NOPs) can abrogate cART efficacy. Here, we aimed to characterize the HIV-1 pol mutation landscape in Cameroon, where highly diverse HIV clades circulate, and identify novel treatment-associated mutations that can potentially affect cART efficacy. More than 8,000 functional Cameroonian HIV-1 pol sequences from 1987 to 2020 were studied for DRMs and NOPs. Site-specific amino acid frequencies and quaternary structural features were determined and compared between periods before (≤2003) and after (2004-2020) regional implementation of cART. cART usage in Cameroon induced deep mutation imprints in reverse transcriptase (RT) and to a lower extent in protease (PR) and integrase (IN), according to their relative usage. In the predominant circulating recombinant form (CRF) 02_AG (CRF02_AG), 27 canonical DRMs and 29 NOPs significantly increased or decreased in RT during cART scale-up, whereas in IN, no DRM and only seven NOPs significantly changed. The profound genomic imprints and higher prevalence of DRMs in RT compared to PR and IN mirror the dominant use of reverse transcriptase inhibitors (RTIs) in sub-Saharan Africa and the predominantly integrase strand transfer inhibitor (InSTI)-naïve study population. Our results support the potential of InSTIs for antiretroviral treatment in Cameroon; however, close surveillance of IN mutations will be required to identify emerging resistance patterns, as observed in RT and PR. Population-wide genomic analyses help reveal the presence of selective pressures and viral adaptation processes to guide strategies to bypass resistance and reinstate effective treatment.