Publications by authors named "Susan Zolla-Pazner"

155 Publications

Quantifying Absolute Neutralization Titers against SARS-CoV-2 by a Standardized Virus Neutralization Assay Allows for Cross-Cohort Comparisons of COVID-19 Sera.

mBio 2021 02 16;12(1). Epub 2021 Feb 16.

Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, USA

The global coronavirus disease 2019 (COVID-19) pandemic has mobilized efforts to develop vaccines and antibody-based therapeutics, including convalescent-phase plasma therapy, that inhibit viral entry by inducing or transferring neutralizing antibodies (nAbs) against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein (CoV2-S). However, rigorous efficacy testing requires extensive screening with live virus under onerous biosafety level 3 (BSL3) conditions, which limits high-throughput screening of patient and vaccine sera. Myriad BSL2-compatible surrogate virus neutralization assays (VNAs) have been developed to overcome this barrier. Yet, there is marked variability between VNAs and how their results are presented, making intergroup comparisons difficult. To address these limitations, we developed a standardized VNA using CoV2-S pseudotyped particles (CoV2pp) based on vesicular stomatitis virus bearing the luciferase gene in place of its G glycoprotein (VSVΔG); this assay can be robustly produced at scale and generate accurate neutralizing titers within 18 h postinfection. Our standardized CoV2pp VNA showed a strong positive correlation with CoV2-S enzyme-linked immunosorbent assay (ELISA) results and live-virus neutralizations in confirmed convalescent-patient sera. Three independent groups subsequently validated our standardized CoV2pp VNA ( > 120). Our data (i) show that absolute 50% inhibitory concentration (absIC), absIC, and absIC values can be legitimately compared across diverse cohorts, (ii) highlight the substantial but consistent variability in neutralization potency across these cohorts, and (iii) support the use of the absIC as a more meaningful metric for assessing the neutralization potency of a vaccine or convalescent-phase sera. Lastly, we used our CoV2pp in a screen to identify ultrapermissive 293T clones that stably express ACE2 or ACE2 plus TMPRSS2. When these are used in combination with our CoV2pp, we can produce CoV2pp sufficient for 150,000 standardized VNAs/week. Vaccines and antibody-based therapeutics like convalescent-phase plasma therapy are premised upon inducing or transferring neutralizing antibodies that inhibit SARS-CoV-2 entry into cells. Virus neutralization assays (VNAs) for measuring neutralizing antibody titers (NATs) are an essential part of determining vaccine or therapeutic efficacy. However, such efficacy testing is limited by the inherent dangers of working with the live virus, which requires specialized high-level biocontainment facilities. We therefore developed a standardized replication-defective pseudotyped particle system that mimics the entry of live SARS-CoV-2. This tool allows for the safe and efficient measurement of NATs, determination of other forms of entry inhibition, and thorough investigation of virus entry mechanisms. Four independent labs across the globe validated our standardized VNA using diverse cohorts. We argue that a standardized and scalable assay is necessary for meaningful comparisons of the myriad of vaccines and antibody-based therapeutics becoming available. Our data provide generalizable metrics for assessing their efficacy.
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http://dx.doi.org/10.1128/mBio.02492-20DOI Listing
February 2021

Signal peptide of HIV-1 envelope modulates glycosylation impacting exposure of V1V2 and other epitopes.

PLoS Pathog 2020 12 28;16(12):e1009185. Epub 2020 Dec 28.

Icahn School of Medicine at Mount Sinai, New York, New York, United States of America.

HIV-1 envelope (Env) is a trimer of gp120-gp41 heterodimers, synthesized from a precursor gp160 that contains an ER-targeting signal peptide (SP) at its amino-terminus. Each trimer is swathed by ~90 N-linked glycans, comprising complex-type and oligomannose-type glycans, which play an important role in determining virus sensitivity to neutralizing antibodies. We previously examined the effects of single point SP mutations on Env properties and functions. Here, we aimed to understand the impact of the SP diversity on glycosylation of virus-derived Env and virus neutralization by swapping SPs. Analyses of site-specific glycans revealed that SP swapping altered Env glycan content and occupancy on multiple N-linked glycosites, including conserved N156 and N160 glycans in the V1V2 region at the Env trimer apex and N88 at the trimer base. Virus neutralization was also affected, especially by antibodies against V1V2, V3, and gp41. Likewise, SP swaps affected the recognition of soluble and cell-associated Env by antibodies targeting distinct V1V2 configurations, V3 crown, and gp41 epitopes. These data highlight the contribution of SP sequence diversity in shaping the Env glycan content and its impact on the configuration and accessibility of V1V2 and other Env epitopes.
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http://dx.doi.org/10.1371/journal.ppat.1009185DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7793277PMC
December 2020

Role of IgM and IgA Antibodies in the Neutralization of SARS-CoV-2.

J Infect Dis 2020 Dec 24. Epub 2020 Dec 24.

Division of Infectious Diseases, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY USA.

Background: SARS-CoV-2 has infected millions of people globally. Virus infection requires the receptor-binding domain (RBD) of the spike protein. Although studies have demonstrated anti-spike and -RBD antibodies to be protective in animal models, and convalescent plasma as a promising therapeutic option, little is known about immunoglobulin (Ig) isotypes capable of blocking infection.

Methods: We studied spike- and RBD-specific Ig isotypes in convalescent and acute plasma/sera using a multiplex bead assay. We also determined virus neutralization activities in plasma, sera, and purified Ig fractions using a VSV pseudovirus assay.

Results: Spike- and RBD-specific IgM, IgG1, and IgA1 were produced by all or nearly all subjects at variable levels and detected early after infection. All samples displayed neutralizing activity. Regression analyses revealed that IgM and IgG1 contributed most to neutralization, consistent with IgM and IgG fractions' neutralization potency. IgA also exhibited neutralizing activity, but with lower potency.

Conclusion: IgG, IgM and IgA are critical components of convalescent plasma used for COVID-19 treatment.
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http://dx.doi.org/10.1093/infdis/jiaa784DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7798948PMC
December 2020

Role of IgM and IgA Antibodies in the Neutralization of SARS-CoV-2.

medRxiv 2020 Nov 2. Epub 2020 Nov 2.

Division of Infectious Diseases, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY USA.

SARS-CoV-2 has infected millions of people globally. Virus infection requires the receptor-binding domain (RBD) of the spike protein. Although studies have demonstrated anti-spike and - RBD antibodies to be protective in animal models and convalescent plasma as a promising therapeutic option, little is known about immunoglobulin (Ig) isotypes capable of blocking infection. Here, we studied spike- and RBD-specific Ig isotypes in convalescent and acute plasma/sera. We also determined virus neutralization activities in plasma/sera, and purified Ig fractions. Spike- and RBD-specific IgM, IgG1, and IgA1 were produced by all or nearly all subjects at variable levels and detected early after infection. All samples also displayed neutralizing activity. Regression analyses revealed that IgM and IgG1 contributed most to neutralization, consistent with IgM and IgG fractions' neutralization potency. However, IgA also exhibited neutralizing activity at a lower potency. Together, IgG, IgM and IgA are critical components of convalescent plasma used for COVID-19 treatment.
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http://dx.doi.org/10.1101/2020.08.18.20177303DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7654883PMC
November 2020

Robust and Specific Secretory IgA Against SARS-CoV-2 Detected in Human Milk.

iScience 2020 Nov 26;23(11):101735. Epub 2020 Oct 26.

Department of Medicine, Division of Infectious Diseases, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, Box 1090, New York, NY 10029, USA.

The SARS-CoV-2 immune response in human milk has not yet been examined, although protecting infants and young children from COVID-19 is critical for limiting community transmission and preventing serious illness and death. Here, milk samples from eight COVID-19-recovered and seven COVID-19-suspected donors were tested for antibody (Ab) binding to the SARS-CoV-2 Spike protein. All samples exhibited significant specific IgA reactivity to the full Spike, whereas 80% exhibited significant IgA and secretory (s)Ab binding to the Receptor-Binding Domain (RBD). Additionally, 67% samples exhibited IgG and/or IgM binding to RBD. IgA and sAb titers were highly correlated, indicating most IgA to be sIgA. Overall, these data indicate that a robust sIgA-dominant SARS-CoV-2 Ab response in human milk after infection should be expected in a significant majority of individuals. Further research is highly warranted to determine Ab functionality and the potential for exploiting extracted milk sIgA for therapeutic use.
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http://dx.doi.org/10.1016/j.isci.2020.101735DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7586930PMC
November 2020

Priming with DNA Expressing Trimeric HIV V1V2 Alters the Immune Hierarchy Favoring the Development of V2-Specific Antibodies in Rhesus Macaques.

J Virol 2020 12 22;95(2). Epub 2020 Dec 22.

Human Retrovirus Pathogenesis Section, Vaccine Branch, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, Maryland, USA

The RV144 vaccine trial revealed a correlation between reduced risk of HIV infection and the level of nonneutralizing-antibody (Ab) responses targeting specific epitopes in the second variable domain (V2) of the HIV gp120 envelope (Env) protein, suggesting this region as a target for vaccine development. To favor induction of V2-specific Abs, we developed a vaccine regimen that included priming with DNA expressing an HIV V1V2 trimeric scaffold immunogen followed by booster immunizations with a combination of DNA and protein in rhesus macaques. Priming vaccination with DNA expressing the HIV recombinant subtype CRF01_AE V1V2 scaffold induced higher and broader V2-specific Ab responses than vaccination with DNA expressing CRF01_AE gp145 Env. Abs recognizing the V2 peptide that was reported as a critical target in RV144 developed only after the priming immunization with V1V2 DNA. The V2-specific Abs showed several nonneutralizing Fc-mediated functions, including ADCP and C1q binding. Importantly, robust V2-specific Abs were maintained upon boosting with gp145 DNA and gp120 protein coimmunization. In conclusion, priming with DNA expressing the trimeric V1V2 scaffold alters the hierarchy of humoral immune responses to V2 region epitopes, providing a method for more efficient induction and maintenance of V2-specific Env Abs associated with reduced risk of HIV infection. The aim of this work was to design and test a vaccine regimen focusing the immune response on targets associated with infection prevention. We demonstrated that priming with a DNA vaccine expressing only the HIV Env V1V2 region induces Ab responses targeting the critical region in V2 associated with protection. This work shows that V1V2 scaffold DNA priming immunization provides a method to focus immune responses to the desired target region, in the absence of immune interference by other epitopes. This induced immune responses with improved recognition of epitopes important for protective immunity, namely, V2-specific humoral immune responses inversely correlating with HIV risk of infection in the RV144 trial.
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http://dx.doi.org/10.1128/JVI.01193-20DOI Listing
December 2020

A High-Throughput Assay for Circulating Antibodies Directed Against the S Protein of Severe Acute Respiratory Syndrome Coronavirus 2.

J Infect Dis 2020 10;222(10):1629-1634

Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, New York, USA.

More than 24 million infections with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were confirmed globally by September 2020. While polymerase chain reaction-based assays are used for diagnosis, there is a need for high-throughput, rapid serologic methods. A Luminex binding assay was developed and used to assess simultaneously the presence of coronavirus disease 2019 (COVID-19)-specific antibodies in human serum and plasma. Clear differentiation was achieved between specimens from infected and uninfected subjects, and a wide range of serum/plasma antibody levels was delineated in infected subjects. All 25 specimens from 18 patients with COVID-19 were positive in the assays with both the trimeric spike and the receptor-binding domain proteins. None of the 13 specimens from uninfected subjects displayed antibodies to either antigen. There was a highly statistically significant difference between the antibody levels of COVID-19-infected and -uninfected specimens (P < .0001). This high-throughput antibody assay is accurate, requires only 2.5 hours, and uses 5 ng of antigen per test.
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http://dx.doi.org/10.1093/infdis/jiaa531DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7499578PMC
October 2020

Quantifying absolute neutralization titers against SARS-CoV-2 by a standardized virus neutralization assay allows for cross-cohort comparisons of COVID-19 sera.

medRxiv 2020 Aug 15. Epub 2020 Aug 15.

Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029.

The on-going coronavirus disease 2019 (COVID-19) pandemic has mobilized a global effort to develop vaccines and therapeutics that inhibit viral entry by inducing or transferring antibodies against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein (CoV2-S). Phase I/II vaccine clinical trials, monoclonal antibodies, and convalescent sera have all shown promise. However, these efforts often require extensive screening with the live virus under onerous high biocontainment conditions (BSL-3). Virus neutralization assays (VNAs) remain the gold standard for evaluating the anti-viral potency of antibodies and entry inhibitors. The proliferation of pseudotyped virus systems that can be used in BSL-2 compatible VNAs is a positive development. Yet, there is marked variability between VNAs and how the findings are presented, making inter-group comparisons difficult. To address these limitations, we developed a standardized VNA using VSVdeltaG based CoV-2-S pseudotyped particles (CoV2pp) that can be robustly produced at scale. We used our CoV2pp to interrogate the role of exogenous and endogenous proteases in CoV-2-S mediated entry and standardized our VNA based on that understanding. Our CoV2pp VNA showed a strong positive correlation with CoV2-S ELISA and live virus neutralizations in a validated set of patient sera. Our system was subsequently validated by three independent groups as an out-of-the-box VNA. More than 120 patient sera were screened, and we report descriptive statistics for absolute (abs) IC50, IC80, and IC90 values from all positive patient sera. Lastly, we used our CoV2pp in a screen to identify ultrapermissive 293T clones that stably express ACE2 or ACE2+TMPRSS2. When used in combination with our CoV2pp, we can now produce CoV2pp sufficient for 150,000 standardized VNA/week.
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http://dx.doi.org/10.1101/2020.08.13.20157222DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7430605PMC
August 2020

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

J Virol 2020 08 17;94(17). Epub 2020 Aug 17.

Department of Medicine, Division of Infectious Diseases, Icahn School of Medicine at Mount Sinai, New York, New York, USA.

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. 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.
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http://dx.doi.org/10.1128/JVI.01175-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7431793PMC
August 2020

A High Through-put Assay for Circulating Antibodies Directed against the S Protein of Severe Acute Respiratory Syndrome Corona virus 2.

medRxiv 2020 Apr 17. Epub 2020 Apr 17.

Division of Infectious Diseases, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY USA.

Background: More than one million infections with the severe acute respiratory syndrome corona virus 2 (SARS-CoV-2) have been confirmed. While PCR-based assays are used for diagnosis, high through-put serologic methods are needed to detect antibodies for seroserveillance and for identification of seroconversion, potential plasma donors, and the nature of the immune response to this pathogen.

Methods: A Luminex binding assay was used to assess the presence of antibodies in human sera from COVID-19-infected and -uninfected individuals specific for two recombinant proteins of SARS-CoV-2.

Findings: Fluorochrome-labeled beads were coated with a recombinant soluble stabilized trimeric SARS-CoV-2 S protein ectodomain or its central portion, the receptor binding domain (RBD). Coated beads were incubated with sera, followed by incubation with biotinylated anti-human total Ig antibodies and phycoerythrin (PE)-labeled streptavidin. Readout using a Luminex analyzer clearly differentiated between sera of the infected and uninfected subject, delineating a wide range of serum antibody levels in infected subjects.

Interpretation: Antibody assays of sera can identify individuals who are infected with SARS-CoV-2 and have seroconverted, as well as subjects who have been infected and recovered. The use of the Luminex binding Ab assay has the advantage that it can be run in approximately 2.5 hours, uses very little antigen, and permits a high through-put of samples/day.

Funding: NIAID contracts and grants, Department of Veterans Affairs grants, the Microbiology Laboratory Clinical Services, Translational Science Hub, and Personalized Virology Initiative, and Department of Medicine of Mount Sinai Health System and Icahn School of Medicine at Mount Sinai.
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http://dx.doi.org/10.1101/2020.04.14.20059501DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7276036PMC
April 2020

Induction of cross-reactive HIV-1 specific antibody responses by engineered V1V2 immunogens with reduced conformational plasticity.

Vaccine 2020 04 17;38(18):3436-3446. Epub 2020 Mar 17.

Thayer School of Engineering, Dartmouth College, Hanover, NH, USA; Department of Microbiology and Immunology, Geisel School of Medicine, Dartmouth College, Hanover, NH, USA. Electronic address:

Antibodies against the HIV-1 V1V2 loops were the only correlate of reduced infection risk in the RV144 vaccine trial, highlighting the V1V2 loops as promising targets for vaccine design. The V1V2 loops are structurally plastic, exhibiting either an α-helix-coil or β-strand conformation. V1V2-specific antibodies may thus recognize distinct conformations, and an antibody's conformational specificity can be an important determinant of breadth and function. Restricting V1V2 conformational plasticity in an immunogen may thus provide control over the conformational specificity and quality of a vaccine-elicited antibody response. Previously, we identified a V1V2 sequence variant (K155M) that results in enhanced recognition by cross-reactive antibodies recognizing the β-strand conformation. Here, we relate V1V2 antigenicity to immunogenicity by comparing the immunogenicity profiles of wildtype and K155M immunogens in two mouse models. In one model, immunization with gp70 V1V2 K155M but not wildtype elicited antibody responses that were cross-reactive to a panel of heterologous gp120 and gp140 antigens. In a second model, we compared the effect of K155M on immunogenicity in the context of gp70 V1V2, gD V1V2 and gp120, examining the effects of scaffold, epitope-focusing and immunization regimen. K155M variants, especially in the context of a gp120 immunogen, resulted in more robust, durable and cross-reactive antibody responses than wildtype immunogens. Restriction of the β-stranded V1V2 conformation in K155M immunogens may thus be associated with the induction of cross-reactive antibody responses thought to be required of a protective HIV-1 vaccine.
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http://dx.doi.org/10.1016/j.vaccine.2020.03.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7132531PMC
April 2020

Multimeric Epitope-Scaffold HIV Vaccines Target V1V2 and Differentially Tune Polyfunctional Antibody Responses.

Cell Rep 2019 07;28(4):877-895.e6

Department of Medicine, Division of Infectious Diseases, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA. Electronic address:

The V1V2 region of the HIV-1 envelope is the target of several broadly neutralizing antibodies (bNAbs). Antibodies to V1V2 elicited in the RV144 clinical trial correlated with a reduced risk of HIV infection, but these antibodies were without broad neutralizing activity. Antibodies targeting V1V2 also correlated with a reduced viral load in immunized macaques challenged with simian immunodeficiency virus (SIV) or simian/human immunodeficiency virus (SHIV). To focus immune responses on V1V2, we engrafted the native, glycosylated V1V2 domain onto five different multimeric scaffold proteins and conducted comparative immunogenicity studies in macaques. Vaccinated macaques developed high titers of plasma and mucosal antibodies that targeted structurally distinct V1V2 epitopes. Plasma antibodies displayed limited neutralizing activity but were functionally active for ADCC and phagocytosis, which was detectable 1-2 years after immunizations ended. This study demonstrates that multivalent, glycosylated V1V2-scaffold protein immunogens focus the antibody response on V1V2 and are differentially effective at inducing polyfunctional antibodies with characteristics associated with protection.
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http://dx.doi.org/10.1016/j.celrep.2019.06.074DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6666430PMC
July 2019

Revisiting the Correlate of Reduced HIV Infection Risk in the Rv144 Vaccine Trial.

J Virol 2019 09 13;93(17). Epub 2019 Aug 13.

Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center and Department of Biostatistics, University of Washington, Seattle, Washington, USA.

The RV144 vaccine trial is the only clinical study to have shown a modest but statistically significant decrease in HIV infection risk. RV144 and the subsequent studies identifying the level of V1V2-specific antibodies as a correlate of reduced infection risk are still controversial despite many papers supporting and expanding the initial study. We address these controversies and summarize active-immunization and passive-immunization experiments in nonhuman primates that support the initial finding.
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http://dx.doi.org/10.1128/JVI.00629-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6694814PMC
September 2019

Immune Correlates of Disease Progression in Linked HIV-1 Infection.

Front Immunol 2019 14;10:1062. Epub 2019 May 14.

Department of Pathology, New York University School of Medicine, New York, NY, United States.

Genetic and immunologic analyses of epidemiologically-linked HIV transmission enable insights into the impact of immune responses on clinical outcomes. Human vaccine trials and animal studies of HIV-1 infection have suggested immune correlates of protection; however, their role in natural infection in terms of protection from disease progression is mostly unknown. Four HIV-1 Cameroonian individuals, three of them epidemiologically-linked in a polygamous heterosexual relationship and one incidence-matched case, were studied over 15 years for heterologous and cross-neutralizing antibody responses, antibody binding, IgA/IgG levels, antibody-dependent cellular cytotoxicity (ADCC) against cells expressing wild-type or CD4-bound Env, viral evolution, Env epitopes, and host factors including HLA-I alleles. Despite viral infection with related strains, the members of the transmission cluster experienced contrasting clinical outcomes including cases of rapid progression and long-term non-progression in the absence of strongly protective HLA-I or CCR5Δ32 alleles. Slower progression and higher CD4/CD8 ratios were associated with enhanced IgG antibody binding to native Env and stronger V1V2 antibody binding responses in the presence of viruses with residue K169 in V2. ADCC against cells expressing Env in the CD4-bound conformation in combination with low Env-specific IgA/IgG ratios correlated with better clinical outcome. This data set highlights for the first time that V1V2-directed antibody responses and ADCC against cells expressing open, CD4-exposed Env, in the presence of low plasma IgA/IgG ratios, can correlate with clinical outcome in natural infection. These parameters are comparable to the major correlates of protection, identified in the RV144 vaccine trial; thus, they may also modulate the rate of clinical progression once infected. The findings illustrate the potential of immune correlate analysis in natural infection to guide vaccine development.
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http://dx.doi.org/10.3389/fimmu.2019.01062DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6527802PMC
August 2020

Vaccine-induced V1V2-specific antibodies control and or protect against infection with HIV, SIV and SHIV.

Curr Opin HIV AIDS 2019 07;14(4):309-317

Division of Infectious Diseases, Department of Medicine, Icahn School of Medicine at Mount Sinai.

Purpose Of Review: In humans, only one independent immunologic correlate of reduced risk of HIV infection has been identified: a robust antibody (Ab) response to the V1V2 domain of the gp120 envelope (Env) protein. In recent years, the presence and level of V1V2-specific Abs has also been correlated with protection from SIV and SHIV infections. Here, we review the multitude of studies showing the in-vivo protective effects of V1V2 Abs and review their immunologic characteristics and antiviral functions.

Recent Findings: Structural and immunologic studies have defined four epitope families in the V1V2 domain: one epitope family, V2q, which preferentially presents as a quaternary structure of the Env trimer, and another epitope family (V2qt) which requires the quaternary trimeric Env structure; these two epitope types are recognized by two families of monoclonal Abs (mAbs)-V2q-specific and V2qt-specific mAbs-which display broad and potent neutralizing activity. A third epitope family, V2i, is present as a discontinuous conformational structure that overlays the α4β7 integrin binding motif, and a fourth epitope family (V2p) exists on V2 peptides. Antibodies specific for V2i and V2p epitopes display only poor neutralizing activity but effectively mediate other antiviral activities and have been correlated with control of and/or protection from HIV, SIV and SHIV. Notably, V2q and V2qt Abs have not been induced by any vaccines, but V2p and V2i Abs have been readily induced with various vaccines in nonhuman primates and humans.

Summary: The correlation of vaccine-induced V2p and V2i Abs with protection from HIV, SIV and SHIV suggests that these Ab types are extremely important to induce with prophylactic vaccines.
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http://dx.doi.org/10.1097/COH.0000000000000551DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6542703PMC
July 2019

Anti-V2 antibody deficiency in individuals infected with HIV-1 in Cameroon.

Virology 2019 03 14;529:57-64. Epub 2019 Jan 14.

Department of Pathology, New York University School of Medicine, New York, NY 10016, USA. Electronic address:

The results of the RV144 vaccine clinical trial showed a correlation between high level of anti-V1V2 antibodies (Abs) and a decreased risk of acquiring HIV-1 infection. This turned the focus of HIV vaccine design to the induction of elevated levels of anti-V2 Abs to increase vaccine efficacy. In plasma samples from HIV-1 infected Cameroonian individuals, we observed broad variations in levels of anti-V2 Abs, and 6 of the 79 plasma samples tested longitudinally displayed substantial deficiency of V2 Abs. Sequence analysis of the V2 region from plasma viruses and multivariate analyses of V2 characteristics showed a significant difference in several features between V2-deficient and V2-reactive plasma Abs. These results suggest that HIV vaccine immunogens containing a shorter V2 region with fewer glycosylation sites and higher electrostatic charges can be beneficial for induction of a higher level of anti-V2 Abs and thus contribute to HIV vaccine efficacy.
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http://dx.doi.org/10.1016/j.virol.2019.01.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6512328PMC
March 2019

Primary Human Neutrophils Exhibit a Unique HIV-Directed Antibody-Dependent Phagocytosis Profile.

J Innate Immun 2019 17;11(2):181-190. Epub 2018 Dec 17.

Department of Infectious Diseases, Icahn School of Medicine at Mount Sinai, New York, New York, USA.

The only clinical HIV vaccine trial to demonstrate efficacy, RV144, correlated protection with the antibodies (Abs) mediating function via the "constant" immunoglobulin region, the crystallizable fragment (Fc). These data have supported a focus on the induction of Abs by vaccines that trigger antiviral activities by relevant leukocytes via Fc receptors (FcRs). Neutrophils are phagocytes that comprise > 50% of leukocytes and display unique FcRs. We sought to compare the Ab-dependent cellular phagocytosis (ADCP) activity of human neutrophils to the commonly assayed THP-1 cell line. HIV-specific Abs were employed to elicit ADCP of beads coated with HIV envelope protein. Overall, trends were noted among neutrophil donors and the ADCP profile was different from that of THP-1 cells. mAb ELISA titers correlated with ADCP by THP-1 cells but not neutrophils. Monoclonal (m)Abs were also tested with primary monocytes. Donor-to-donor variation was high, and hindered the analysis of this dataset, but it was, in itself, an important finding. This study illustrates the concept that the assessment of FcR-mediated Ab activity with a frequently used cell line such as THP-1 is not necessarily indicative of relevant Ab functionality in vivo, and this calls for in-depth study of the properties of the HIV antibodies best-suited to eliciting antiviral activities by primary cells.
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http://dx.doi.org/10.1159/000494371DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6402977PMC
May 2020

Modulation of Antibody Responses to the V1V2 and V3 Regions of HIV-1 Envelope by Immune Complex Vaccines.

Front Immunol 2018 26;9:2441. Epub 2018 Oct 26.

Icahn School of Medicine at Mount Sinai, New York, NY, United States.

Prophylactic HIV vaccines must elicit antibodies (Abs) against the virus envelope glycoproteins (Env) to effectively prevent HIV infection. We investigated a vaccine platform that utilizes immune complexes made of Env proteins gp120 and monoclonal Abs (mAbs) against different gp120 epitopes. We previously observed alterations in V3 antigenicity upon formation of certain gp120/mAb complexes and demonstrated the ability of these complexes to modulate the elicitation of V3 Ab responses. However, the effects on the V1V2 domain, an important target for Abs that correlate with vaccine-induced protection against HIV, have not been studied, nor have immune complex vaccines made with non-B subtype Env. This study compared subtypes B (JRFL) and CRF_01.AE (A244) Env gp120 proteins in complex with selected gp120-specific mAbs. Allosteric and antigenic changes were detected on these immune complexes, indicating that gp120/mAb interaction induces alterations on the Env surface that may modify the Env immunogenic properties. To evaluate this idea, mice were immunized with gp120/mAb complexes or their uncomplexed gp120 counterparts. The overall serum IgG titers elicited against gp120 were comparable, but a marked skewing toward V1V2 or V3 was evident and dependent on the gp120 strain and the specificity of the mAb used to form the complexes. Compared with uncomplexed gp120, gp120 complexed with CD4bs or V1V2 mAbs, but not with C2 or V3 mAbs, elicited V3 Abs of greater titers and breadth, and Abs more capable of neutralizing tier 1 virus. Epitope mapping revealed a shift to a more conserved site in the V3 crown. However, the complexes did not enhance V1V2 Ab response, and the elicited V1V2 Abs were not cross-reactive. This profile contrasts with Ab responses to gp120/mAb complexes. Notably, gp120/mAb complexes induced higher levels of V1V2 Abs with some cross-reactivity, while also stimulating weak or strain-specific V3 Abs. Sera from gp120/mAb complex-immunized animals displayed no measurable virus neutralization but did mediate Ab-dependent cellular phagocytosis, albeit at levels similar to that induced by gp120 alone. These data indicate the potential utility of immune complexes as vaccines to shape Ab responses toward or away from Env sites of interest.
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http://dx.doi.org/10.3389/fimmu.2018.02441DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6212562PMC
September 2019

Generation and characterization of a bivalent protein boost for future clinical trials: HIV-1 subtypes CR01_AE and B gp120 antigens with a potent adjuvant.

PLoS One 2018 26;13(4):e0194266. Epub 2018 Apr 26.

GSK, Rockville, MD, United States of America.

The RV144 Phase III clinical trial with ALVAC-HIV prime and AIDSVAX B/E subtypes CRF01_AE (A244) and B (MN) gp120 boost vaccine regime in Thailand provided a foundation for the future development of improved vaccine strategies that may afford protection against the human immunodeficiency virus type 1 (HIV-1). Results from this trial showed that immune responses directed against specific regions V1V2 of the viral envelope (Env) glycoprotein gp120 of HIV-1, were inversely correlated to the risk of HIV-1 infection. Due to the low production of gp120 proteins in CHO cells (2-20 mg/L), cleavage sites in V1V2 loops (A244) and V3 loop (MN) causing heterogeneous antigen products, it was an urgent need to generate CHO cells harboring A244 gp120 with high production yields and an additional, homogenous and uncleaved subtype B gp120 protein to replace MN used in RV144 for the future clinical trials. Here we describe the generation of Chinese Hamster Ovary (CHO) cell lines stably expressing vaccine HIV-1 Env antigens for these purposes: one expressing an HIV-1 subtype CRF01_AE A244 Env gp120 protein (A244.AE) and one expressing an HIV-1 subtype B 6240 Env gp120 protein (6240.B) suitable for possible future manufacturing of Phase I clinical trial materials with cell culture expression levels of over 100 mg/L. The antigenic profiles of the molecules were elucidated by comprehensive approaches including analysis with a panel of well-characterized monoclonal antibodies recognizing critical epitopes using Biacore and ELISA, and glycosylation analysis by mass spectrometry, which confirmed previously identified glycosylation sites and revealed unknown sites of O-linked and N-linked glycosylations at non-consensus motifs. Overall, the vaccines given with MF59 adjuvant induced higher and more rapid antibody (Ab) responses as well as higher Ab avidity than groups given with aluminum hydroxide. Also, bivalent proteins (A244.AE and 6240.B) formulated with MF59 elicited distinct V2-specific Abs to the epitope previously shown to correlate with decreased risk of HIV-1 infection in the RV144 trial. All together, these results provide critical information allowing the consideration of these candidate gp120 proteins for future clinical evaluations in combination with a potent adjuvant.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0194266PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5919662PMC
July 2018

Fine epitope signature of antibody neutralization breadth at the HIV-1 envelope CD4-binding site.

JCI Insight 2018 03 8;3(5). Epub 2018 Mar 8.

Thayer School of Engineering and.

Major advances in donor identification, antigen probe design, and experimental methods to clone pathogen-specific antibodies have led to an exponential growth in the number of newly characterized broadly neutralizing antibodies (bnAbs) that recognize the HIV-1 envelope glycoprotein. Characterization of these bnAbs has defined new epitopes and novel modes of recognition that can result in potent neutralization of HIV-1. However, the translation of envelope recognition profiles in biophysical assays into an understanding of in vivo activity has lagged behind, and identification of subjects and mAbs with potent antiviral activity has remained reliant on empirical evaluation of neutralization potency and breadth. To begin to address this discrepancy between recombinant protein recognition and virus neutralization, we studied the fine epitope specificity of a panel of CD4-binding site (CD4bs) antibodies to define the molecular recognition features of functionally potent humoral responses targeting the HIV-1 envelope site bound by CD4. Whereas previous studies have used neutralization data and machine-learning methods to provide epitope maps, here, this approach was reversed, demonstrating that simple binding assays of fine epitope specificity can prospectively identify broadly neutralizing CD4bs-specific mAbs. Building on this result, we show that epitope mapping and prediction of neutralization breadth can also be accomplished in the assessment of polyclonal serum responses. Thus, this study identifies a set of CD4bs bnAb signature amino acid residues and demonstrates that sensitivity to mutations at signature positions is sufficient to predict neutralization breadth of polyclonal sera with a high degree of accuracy across cohorts and across clades.
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http://dx.doi.org/10.1172/jci.insight.97018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5922287PMC
March 2018

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.

J Virol 2018 06 14;92(11). Epub 2018 May 14.

Oregon National Primate Research Center, Oregon Health & Science University, Portland, Oregon, USA

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 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 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. 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 that could contribute to a protective HIV vaccine.
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http://dx.doi.org/10.1128/JVI.02198-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5952134PMC
June 2018

HIV-1 Envelope Glycoproteins from Diverse Clades Differentiate Antibody Responses and Durability among Vaccinees.

J Virol 2018 04 28;92(8). Epub 2018 Mar 28.

Duke Human Vaccine Institute, Duke University School of Medicine, Durham, North Carolina, USA

Induction of broadly cross-reactive antiviral humoral responses with the capacity to target globally diverse circulating strains is a key goal for HIV-1 immunogen design. A major gap in the field is the identification of diverse HIV-1 envelope antigens to evaluate vaccine regimens for binding antibody breadth. In this study, we define unique antigen panels to map HIV-1 vaccine-elicited antibody breadth and durability. Diverse HIV-1 envelope glycoproteins were selected based on genetic and geographic diversity to cover the global epidemic, with a focus on sexually acquired transmitted/founder viruses with a tier 2 neutralization phenotype. Unique antigenicity was determined by nonredundancy (Spearman correlation), and antigens were clustered using partitioning around medoids (PAM) to identify antigen diversity. Cross-validation demonstrated that the PAM method was better than selection by reactivity and random selection. Analysis of vaccine-elicited V1V2 binding antibody in longitudinal samples from the RV144 clinical trial revealed the striking heterogeneity among individual vaccinees in maintaining durable responses. These data support the idea that a major goal for vaccine development is to improve antibody levels, breadth, and durability at the population level. Elucidating the level and durability of vaccine-elicited binding antibody breadth needed for protection is critical for the development of a globally efficacious HIV vaccine. The path toward an efficacious HIV-1 vaccine will require characterization of vaccine-induced immunity that can recognize and target the highly genetically diverse virus envelope glycoproteins. Antibodies that target the envelope glycoproteins, including diverse sequences within the first and second hypervariable regions (V1V2) of gp120, were identified as correlates of risk for the one partially efficacious HIV-1 vaccine. To build upon this discovery, we experimentally and computationally evaluated humoral responses to define envelope glycoproteins representative of the antigenic diversity of HIV globally. These diverse envelope antigens distinguished binding antibody breadth and durability among vaccine candidates, thus providing insights for advancing the most promising HIV-1 vaccine candidates.
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http://dx.doi.org/10.1128/JVI.01843-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5874409PMC
April 2018

Alterations of HIV-1 envelope phenotype and antibody-mediated neutralization by signal peptide mutations.

PLoS Pathog 2018 01 25;14(1):e1006812. Epub 2018 Jan 25.

Icahn School of Medicine at Mount Sinai, Division of Infectious Diseases, New York, New York, United States of America.

HIV-1 envelope glycoprotein (Env) mediates virus attachment and entry into the host cells. Like other membrane-bound and secreted proteins, HIV-1 Env contains at its N terminus a signal peptide (SP) that directs the nascent Env to the endoplasmic reticulum (ER) where Env synthesis and post-translational modifications take place. SP is cleaved during Env biosynthesis but potentially influences the phenotypic traits of the Env protein. The Env SP sequences of HIV-1 isolates display high sequence variability, and the significance of such variability is unclear. We postulate that changes in the Env SP influence Env transport through the ER-Golgi secretory pathway and Env folding and/or glycosylation that impact on Env incorporation into virions, receptor binding and antibody recognition. We first evaluated the consequences of mutating the charged residues in the Env SP in the context of infectious molecular clone HIV-1 REJO.c/2864. Results show that three different mutations affecting histidine at position 12 affected Env incorporation into virions that correlated with reduction of virus infectivity and DC-SIGN-mediated virus capture and transmission. Mutations at positions 8, 12, and 15 also rendered the virus more resistant to neutralization by monoclonal antibodies against the Env V1V2 region. These mutations affected the oligosaccharide composition of N-glycans as shown by changes in Env reactivity with specific lectins and by mass spectrometry. Increased neutralization resistance and N-glycan composition changes were also observed when analogous mutations were introduced to another HIV-1 strain, JRFL. To the best of our knowledge, this is the first study showing that certain residues in the HIV-1 Env SP can affect virus neutralization sensitivity by modulating oligosaccharide moieties on the Env N-glycans. The HIV-1 Env SP sequences thus may be under selective pressure to balance virus infectiousness with virus resistance to the host antibody responses. (289 words).
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http://dx.doi.org/10.1371/journal.ppat.1006812DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5800646PMC
January 2018

Functional Antibody Response Against V1V2 and V3 of HIV gp120 in the VAX003 and VAX004 Vaccine Trials.

Sci Rep 2018 01 11;8(1):542. Epub 2018 Jan 11.

Department of Medicine, Division of Infectious Diseases, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA.

Immunization with HIV AIDSVAX gp120 vaccines in the phase III VAX003 and VAX004 trials did not confer protection. To understand the shortcomings in antibody (Ab) responses induced by these vaccines, we evaluated the kinetics of Ab responses to the V1V2 and V3 regions of gp120 and the induction of Ab-mediated antiviral functions during the course of 7 vaccinations over a 30.5-month period. Plasma samples from VAX003 and VAX004 vaccinees and placebo recipients were measured for ELISA-binding Abs and for virus neutralization, Ab-dependent cellular phagocytosis (ADCP), and Ab-dependent cellular cytotoxicity (ADCC). Ab responses to V1V2 and V3 peaked after 3 to 4 immunizations and declined after 5 to 7 immunizations. The deteriorating responses were most evident against epitopes in the underside of the V1V2 β-barrel and in the V3 crown. Correspondingly, vaccinees demonstrated higher neutralization against SF162 pseudovirus sensitive to anti-V1V2 and anti-V3 Abs after 3 or 4 immunizations than after 7 immunizations. Higher levels of ADCP and ADCC were also observed at early or mid-time points as compared with the final time point. Hence, VAX003 and VAX004 vaccinees generated V1V2- and V3-binding Abs and functional Abs after 3 to 4 immunizations, but subsequent boosts did not maintain these responses.
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http://dx.doi.org/10.1038/s41598-017-18863-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5765017PMC
January 2018

Non-neutralizing Antibodies Targeting the V1V2 Domain of HIV Exhibit Strong Antibody-Dependent Cell-mediated Cytotoxic Activity.

Sci Rep 2017 10 4;7(1):12655. Epub 2017 Oct 4.

INSERM U1109, Fédération de Médecine Translationnelle de Strasbourg (FMTS), Université de Strasbourg, Strasbourg, France.

The development of an effective vaccine against HIV-1 has proven to be challenging. Broadly neutralizing antibodies (bNAbs), whilst exhibiting neutralization breadth and potency, are elicited only in a small subset of infected individuals and have yet to be induced by vaccination. Case-control studies of RV144 identified an inverse correlation of HIV-1 infection risk with antibodies (Abs) to the V1V2 region of gp120 with high antibody-dependent cellular cytotoxicity (ADCC) activity. The neutralizing activity of Abs was not found to contribute to this protective outcome. Using primary effector and target cells and primary virus isolates, we studied the ADCC profile of different monoclonal Abs targeting the V1V2 loop of gp120 that had low or no neutralizing activity. We compared their ADCC activity to some bNAbs targeting different regions of gp120. We found that mAbs targeting the V1V2 domain induce up to 60% NK cell mediated lysis of HIV-1 infected PBMCs in a physiologically relevant ADCC model, highlighting the interest in inducing such Abs in future HIV vaccine trials. Our data also suggest that in addition to neutralization, lysis of infected cells by Abs can effectively participate in HIV protection, as suggested by the RV144 immune correlate analysis.
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http://dx.doi.org/10.1038/s41598-017-12883-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5627290PMC
October 2017

Non-neutralizing Antibodies Alter the Course of HIV-1 Infection In Vivo.

Cell 2017 Aug 27;170(4):637-648.e10. Epub 2017 Jul 27.

Laboratory of Molecular Immunology, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA; Howard Hughes Medical Institute, The Rockefeller University, 1230 York Ave, New York, NY 10065, USA. Electronic address:

Non-neutralizing antibodies (nnAbs) to HIV-1 show little measurable activity in prevention or therapy in animal models yet were the only correlate of protection in the RV144 vaccine trial. To investigate the role of nnAbs on HIV-1 infection in vivo, we devised a replication-competent HIV-1 reporter virus that expresses a heterologous HA-tag on the surface of infected cells and virions. Anti-HA antibodies bind to, but do not neutralize, the reporter virus in vitro. However, anti-HA protects against infection in humanized mice and strongly selects for nnAb-resistant viruses in an entirely Fc-dependent manner. Similar results were also obtained with tier 2 HIV-1 viruses using a human anti-gp41 nnAb, 246D. While nnAbs are demonstrably less effective than broadly neutralizing antibodies (bNAbs) against HIV-1 in vitro and in vivo, the data show that nnAbs can protect against and alter the course of HIV-1 infection in vivo. PAPERCLIP.
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http://dx.doi.org/10.1016/j.cell.2017.06.048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5554461PMC
August 2017

Plasticity and Epitope Exposure of the HIV-1 Envelope Trimer.

J Virol 2017 09 10;91(17). Epub 2017 Aug 10.

Department of Medicine, Division of Infectious Diseases, Icahn School of Medicine at Mount Sinai, New York, New York, USA.

We recently showed that mutations in the HIV-1 envelope (Env) destabilize the V3 loop, rendering neutralization-resistant viruses sensitive to V3-directed monoclonal antibodies (MAbs). Here, we investigated the propagation of this effect on other Env epitopes, with special emphasis on V2 loop exposure. Wild-type JR-FL and 19 mutant JR-FL pseudoviruses were tested for neutralization sensitivity to 21 MAbs specific for epitopes in V2, the CD4 binding site (CD4bs), and the CD4-induced (CD4i) region. Certain glycan mutants, mutations in the gp120 hydrophobic core, and mutations in residues involved in intraprotomer interactions exposed epitopes in the V2i region (which overlies the α4β7 integrin binding site) and the V3 crown, suggesting general destabilization of the distal region of the trimer apex. In contrast, other glycan mutants, mutations affecting interprotomer interactions, and mutations affecting the CD4bs exposed V3 but not V2i epitopes. These data indicate for the first time that V3 can move independently of V2, with V3 pivoting out from its "tucked" position in the trimer while apparently leaving the V2 apex intact. Notably, none of the mutations exposed V2 epitopes without also exposing V3, suggesting that movement of V2 releases V3. Most mutations increased sensitivity to CD4bs-directed MAbs without exposure of the CD4i epitope, implying these mutations facilitate the trimers' maintenance of an intermediate energy state between open and closed conformations. Taken together, these data indicate that several transient Env epitopes can be rendered more accessible to antibodies (Abs) via specific mutations, and this may facilitate the design of V1V2-targeting immunogens. Many epitopes of the HIV envelope (Env) spike are relatively inaccessible to antibodies (Abs) compared to their exposure in the open Env conformation induced by receptor binding. However, the reduced infection rate that resulted from the vaccine used in the RV144 HIV-1 vaccine trial was correlated with the elicitation of V2- and V3-directed antibodies. Previously, we identified various mechanisms responsible for destabilizing the V3 loop; here, we determined, via mutation of numerous Env residues, which of these elements maintain the V1V2 loop in an inaccessible state and which expose V1V2 and/or V3 epitopes. Notably, our data indicate that V3 can move independently of V2, but none of the mutations studied expose V2 epitopes without also exposing V3. Additionally, V1V2 can be rendered more accessible to Abs via specific mutations, facilitating the development of engineered V2 immunogens.
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http://dx.doi.org/10.1128/JVI.00410-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5553165PMC
September 2017

Differential induction of anti-V3 crown antibodies with cradle- and ladle-binding modes in response to HIV-1 envelope vaccination.

Vaccine 2017 03 6;35(10):1464-1473. Epub 2017 Feb 6.

Department of Medicine, Division of Infectious Diseases, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; James J. Peters VA Medical Center, Bronx, NY 10468, USA. Electronic address:

The V3 loop in the HIV envelope gp120 is one of the immunogenic sites targeted by Abs. The V3 crown in particular has conserved structural elements recognized by cross-reactive neutralizing Abs, indicating its potential contribution in protection against HIV. Crystallographic analyses of anti-V3 crown mAbs in complex with the V3 peptides have revealed that these mAbs recognize the conserved sites on the V3 crown via two distinct strategies: a cradle-binding mode (V3C) and a ladle-binding (V3L) mode. However, almost all of the anti-V3 crown mAbs studied in the past were isolated from chronically HIV-infected individuals. The extents to which the two types of anti-V3 crown Abs are generated by vaccination are unknown. This study analyzed the prevalence of V3C-type and V3L-type Ab responses in HIV-infected individuals and in HIV envelope-immunized humans and animals using peptide mimotopes that distinguish the two Ab types. The results show that both V3L-type and V3C-type Abs were generated by the vast majority of chronically HIV-infected humans, although the V3L-type were more prevalent. In contrast, only one of the two V3 Ab types was elicited in vaccinated humans or animal models, irrespective of HIV-1 envelope clades, envelope constructs (oligomeric or monomeric), and protocols (DNA plus protein or protein alone) used for vaccinations. The V3C-type Abs were produced by vaccinated humans, macaques, and rabbits, whereas the V3L-type Abs were made by mice. The V3C-type and V3L-type Abs generated by the vaccinations were able to mediate virus neutralization. These data indicate the restricted repertoires and the species-specific differences in the functional V3-specific Ab responses induced by the HIV envelope vaccines. The study implies the need for improving immunogen designs and vaccination strategies to broaden the diversity of Abs in order to target the different conserved epitopes in the V3 loop and, by extension, in the entire HIV envelope.
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http://dx.doi.org/10.1016/j.vaccine.2016.11.107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5343672PMC
March 2017

Monoclonal Antibodies Specific for the V2, V3, CD4-Binding Site, and gp41 of HIV-1 Mediate Phagocytosis in a Dose-Dependent Manner.

J Virol 2017 04 29;91(8). Epub 2017 Mar 29.

Department of Pathology, New York University School of Medicine, New York, New York, USA

In light of the weak or absent neutralizing activity mediated by anti-V2 monoclonal antibodies (MAbs), we tested whether they can mediate Ab-dependent cellular phagocytosis (ADCP), which is an important element of anti-HIV-1 immunity. We tested six anti-V2 MAbs and compared them with 21 MAbs specific for V3, the CD4-binding site (CD4bs), and gp41 derived from chronically HIV-1-infected individuals and produced by hybridoma cells. ADCP activity was measured by flow cytometry using uptake by THP-1 monocytic cells of fluorescent beads coated with gp120, gp41, BG505 SOSIP.664, or BG505 DS-SOSIP.664 complexed with MAbs. The measurement of ADCP activity by the area under the curve showed significantly higher activity of anti-gp41 MAbs than of the members of the three other groups of MAbs tested using beads coated with monomeric gp41 or gp120; anti-V2 MAbs were dominant compared to anti-V3 and anti-CD4bs MAbs against clade C gp120 ADCP activity mediated by V2 and V3 MAbs was positive against stabilized DS-SOSIP.664 trimer but negligible against SOSIP.664 targets, suggesting that a closed envelope conformation better exposes the variable loops. Two IgG3 MAbs against the V2 and V3 regions displayed dominant ADCP activity compared to a panel of IgG1 MAbs. This superior ADCP activity was confirmed when two of three recombinant IgG3 anti-V2 MAbs were compared to their IgG1 counterparts. The study demonstrated dominant ADCP activity of anti-gp41 against monomers but not trimers, with some higher activity of anti-V2 MAbs than of anti-V3 and anti-CD4bs MAbs. The ability to mediate ADCP suggests a mechanism by which anti-HIV-1 envelope Abs can contribute to protective efficacy. Anti-V2 antibodies (Abs) correlated with reduced risk of HIV-1 infection in recipients of the RV144 vaccine, suggesting that they play a protective role, but a mechanism providing such protection remains to be determined. The rare and weak neutralizing activities of anti-V2 MAbs prompted us to study Fc-mediated activities. We compared anti-V2 MAbs with other MAbs specific for V3, CD4bs, and gp41 for Ab-dependent cellular phagocytosis (ADCP) activity, implicated in protective immunity. The anti-V2 MAbs displayed stronger activity than other anti-gp120 MAbs in screening against one of two gp120s and against DS-SOSIP, which mimics the native trimer. The activity of anti-gp41 MAbs was superior in targeting monomeric gp41 but was comparable to that seen against trimers, which may not adequately expose gp41 epitopes. While anti-envelope MAbs in general mediated ADCP activity, anti-V2 MAbs displayed some dominance compared to other MAbs. Our demonstration that anti-V2 MAbs mediate ADCP activity suggests a functional mechanism for their contribution to protective efficacy.
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http://dx.doi.org/10.1128/JVI.02325-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5375680PMC
April 2017

Rationally Designed Immunogens Targeting HIV-1 gp120 V1V2 Induce Distinct Conformation-Specific Antibody Responses in Rabbits.

J Virol 2016 Dec 28;90(24):11007-11019. Epub 2016 Nov 28.

Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, New York, USA

The V1V2 region of HIV-1 gp120 harbors a major vulnerable site targeted by a group of broadly neutralizing monoclonal antibodies (MAbs) such as PG9 through strand-strand recognition. However, this epitope region is structurally polymorphic as it can also form a helical conformation recognized by RV144 vaccine-induced MAb CH58. This structural polymorphism is a potential mechanism for masking the V1V2 vulnerable site. Designing immunogens that can induce conformation-specific antibody (Ab) responses may lead to vaccines targeting this vulnerable site. We designed a panel of immunogens engrafting the V1V2 domain into trimeric and pentameric scaffolds in structurally constrained conformations. We also fused V1V2 to an Fc fragment to mimic the unconstrained V1V2 conformation. We tested these V1V2-scaffold proteins for immunogenicity in rabbits and assessed the responses by enzyme-linked immunosorbent assay (ELISA) and competition assays. Our V1V2 immunogens induced distinct conformation-specific Ab responses. Abs induced by structurally unconstrained immunogens reacted preferentially with unconstrained V1V2 antigens, suggesting recognition of the helical configuration, while Abs induced by the structurally constrained immunogens reacted preferentially with constrained V1V2 antigens, suggesting recognition of the β-strand conformation. The Ab responses induced by the structurally constrained immunogens were more broadly reactive and had higher titers than those induced by the structurally unconstrained immunogens. Our results demonstrate that immunogens presenting the different structural conformations of the gp120 V1V2 vulnerable site can be designed and that these immunogens induce distinct Ab responses with epitope conformation specificity. Therefore, these structurally constrained V1V2 immunogens are vaccine prototypes targeting the V1V2 domain of the HIV-1 envelope.

Importance: The correlates analysis of the RV144 HIV-1 vaccine trial suggested that the presence of antibodies to the V1V2 region of HIV-1 gp120 was responsible for the modest protection observed in the trial. In addition, V1V2 harbors one of the key vulnerable sites of HIV-1 Env recognized by a family of broadly neutralizing MAbs such as PG9. Thus, V1V2 is a key target for vaccine development. However, this vulnerable site is structurally polymorphic, and designing immunogens that present different conformations is crucial for targeting this site. We show here that such immunogens can be designed and that they induced conformation-specific antibody responses in rabbits. Our immunogens are therefore prototypes of vaccine candidates targeting the V1V2 region of HIV-1 Env.
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http://dx.doi.org/10.1128/JVI.01409-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5126360PMC
December 2016