Ancestral SARS-CoV-2-Driven Antibody Repertoire Diversity in an Unvaccinated Individual Correlates with Expanded Neutralization Breadth.

Understanding the quality of immune repertoire triggered during natural infection can provide vital clues that form the basis for development of a humoral immune response in some individuals capable of broadly neutralizing pan-SARS-CoV-2 variants. In the present study, we report variations in neutralization potential against Omicron variants of two novel neutralizing monoclonal antibodies (MAbs), THSC20.HVTR11 and THSC20.HVTR55, isolated from an unvaccinated convalescent individual that represent distinct B cell lineage origins and epitope specificity compared to five MAbs we previously reported that were isolated from the same individual. In addition, we observed neutralization of Omicron variants by plasma antibodies obtained from this particular individual postvaccination with increased magnitude. Interestingly, this observation was found to be comparable with six additional individuals who initially were also infected with ancestral SARS-CoV-2 and then received vaccines, indicating that hybrid immunity can provide robust humoral immunity likely by antibody affinity maturation. Development of a distinct antigen-specific B cell repertoire capable of producing polyclonal antibodies with distinct affinity and specificities offers the highest probability of protecting against evolving SARS-CoV-2 variants. IMPORTANCE Development of robust neutralizing antibodies in SARS-CoV-2 convalescent individuals is known; however, it varies at the population level. We isolated monoclonal antibodies from an individual infected with ancestral SARS-CoV-2 in early 2020 that not only varied in their B cell lineage origin but also varied in their capability and potency to neutralize all the known variants of concern (VOCs) and currently circulating Omicron variants. This indicated establishment of unique lineages that contributed in forming a B cell repertoire in this particular individual immediately following infection, giving rise to diverse antibody responses that could complement each other in providing a broadly neutralizing polyclonal antibody response. Individuals who were able to produce polyclonal antibody responses with higher magnitude have a higher chance of being protected from evolving SARS-CoV-2 variants.

A combination of potently neutralizing monoclonal antibodies isolated from an Indian convalescent donor protects against the SARS-CoV-2 Delta variant.

Although efficacious vaccines have significantly reduced the morbidity and mortality of COVID-19, there remains an unmet medical need for treatment options, which monoclonal antibodies (mAbs) can potentially fill. This unmet need is exacerbated by the emergence and spread of SARS-CoV-2 variants of concern (VOCs) that have shown some resistance to vaccine responses. Here we report the isolation of five neutralizing mAbs from an Indian convalescent donor, out of which two (THSC20.HVTR04 and THSC20.HVTR26) showed potent neutralization of SARS-CoV-2 VOCs at picomolar concentrations, including the Delta variant (B.1.617.2). One of these (THSC20.HVTR26) also retained activity against the Omicron variant. These two mAbs target non-overlapping epitopes on the receptor-binding domain (RBD) of the spike protein and prevent virus attachment to its host receptor, human angiotensin converting enzyme-2 (hACE2). Furthermore, the mAb cocktail demonstrated protection against the Delta variant at low antibody doses when passively administered in the K18 hACE2 transgenic mice model, highlighting their potential as a cocktail for prophylactic and therapeutic applications. Developing the capacity to rapidly discover and develop mAbs effective against highly transmissible pathogens like coronaviruses at a local level, especially in a low- and middle-income country (LMIC) such as India, will enable prompt responses to future pandemics as an important component of global pandemic preparedness.

HIV Prevention in a Time of COVID-19: A Report from the HIVR4P // Virtual Conference 2021.

The HIV Research for Prevention (HIVR4P) conference catalyzes knowledge sharing on biomedical HIV prevention interventions such as HIV vaccines, antibody infusions, pre-exposure prophylaxis, and microbicides in totality-from the molecular details and delivery formulations to the behavioral, social, and structural underpinnings. HIVR4P // Virtual was held over the course of 2 weeks on January 27-28 and February 3-4, 2021 as the coronavirus disease 2019 (COVID-19) pandemic continued to inflict unprecedented harm globally. The HIVR4P community came together with 1,802 researchers, care providers, policymakers, implementers, and advocates from 92 countries whose expertise spanned the breadth of the HIV prevention pipeline from preclinical to implementation. The program included 113 oral and 266 poster presentations. This article presents a brief summary of the conference highlights. Complete abstracts, webcasts, and daily rapporteur summaries may be found on the conference website (https://www.hivr4p.org/).

Geospatial HIV-1 subtype C gp120 sequence diversity and its predicted impact on broadly neutralizing antibody sensitivity.

Evolving diversity in globally circulating HIV-1 subtypes presents a formidable challenge in defining and developing neutralizing antibodies for prevention and treatment. HIV-1 subtype C is responsible for majority of global HIV-1 infections. In the present study, we examined the diversity in genetic signatures and attributes that differentiate region-specific HIV-1 subtype C gp120 sequences associated with virus neutralization outcomes to key bnAbs having distinct epitope specificities. A total of 1814 full length HIV-1 subtype C gp120 sequence from 37 countries were retrieved from Los Alamos National Laboratory HIV database (www.hiv.lanl.gov). The amino acid sequences were assessed for their phylogenetic association, variable loop lengths and prevalence of potential N-linked glycosylation sites (pNLGS). Responses of these sequences to bnAbs were predicted with a machine learning algorithm 'bNAb-ReP' and compared with those reported in the CATNAP database. Subtype C sequences from Asian countries including India differed phylogenetically when compared with that from African countries. Variable loop lengths and charges within Indian and African clusters were also found to be distinct from each other, specifically for V1, V2 and V4 loops. Pairwise analyses at each of the 25 pNLG sites indicated distinct country specific profiles. Highly significant differences (p<0.001***) were observed in prevalence of four pNLGS (N130, N295, N392 and N448) between South Africa and India, having most disease burden associated with subtype C. Our findings highlight that distinctly evolving clusters within global intra-subtype C gp120 sequences are likely to influence the disparate region-specific sensitivity of circulating HIV-1 subtype C to bnAbs.

Neutralization diversity of HIV-1 Indian subtype C envelopes obtained from cross sectional and followed up individuals against broadly neutralizing monoclonal antibodies having distinct gp120 specificities.

BACKGROUND: The potential use of the broadly neutralizing monoclonal antibodies (bnAbs) towards prophylaxis and treatment to HIV-1 is currently being explored. While a number of promising bnAbs have been discovered and a few of them have progressed towards clinical development, their extent of neutralization coverage with respect to global HIV-1 variants given the existence of genetically distinct subtypes and recombinants circulating globally is not clearly known. In the present study, we examined the variation in the neutralization susceptibility of pseudoviruses expressing 71 full length primary HIV-1 subtype C envs obtained from limited cross-sectional individuals over different time points against four bnAbs that target gp120 with distinct specificities: VRC01, CAP256-VRC26.25, PGDM1400 and PGT121. RESULTS: We found significant variations in the susceptibility of Indian clade C to these four bnAbs. These variations were found to be distinct to that observed in African subtype C based on the existing datasets and concordant with their sequence diversity. Trend analysis indicated an increasing neutralization resistance observed over time with CAP25-VRC26.25, PGDM1400 and PGT121 when tested on pseudoviruses expressing envs obtained from 1999 to 2016. However, inconsistent trend in neutralization susceptibility was observed, when pseudoviruses expressing envs obtained from three followed up individuals were examined. Finally, through predictive analysis of the 98 Indian subtype C including those assessed in the present study by employing additive model implemented in CombiNAber ( http://www.hiv.lanl.gov ), we observed two possibilities where combinations of three bnAbs (VRC01/CAP56-VRC26.25/PGT121 and PGDM1400/CAP256-VRC26.25/PGT121) could achieve near 100% neutralization coverage. CONCLUSIONS: Our findings not only indicate disparate intra-clade C genetic vis-à-vis neutralization diversities but also warrant the need for more comprehensive study using additional isolates towards comparing inter and intra-clade neutralization diversities which will be necessary for selecting the bnAb combinations suitable for optimal coverage of the region-specific HIV-1 circulating subtypes. Expanding these efforts is imperative for designing efficacious bnAb based intervention strategies for India as well as subtype C in general.

Analysis of sequence diversity and selection pressure in HIV-1 clade C gp41 from India.

Evaluation of viral diversity is critical for the rational design of treatment modalities against Human immunodeficiency virus (HIV). Predominated by HIV-1 clade C (HIV-1C), the epidemic in India represents the third largest population infected with HIV-1 globally. Glycoprotein 41 (gp41) is critical for viral replication and is a target for the design of therapeutic strategies. However, documentation of viral diversity of gp41 gene in infected individuals from India remains limited. Present study employed high throughput sequencing to examine variation in gp41 amplicons generated from blood derived viruses in 24 HIV-1C infected individuals from Mumbai, India. Sequence diversity profiles were documented in different functional domains of gp41. Furthermore, through a meta-analysis approach, all reported gp41 sequences from India (N = 70) were compared with those from South Africa (N = 126), country with the largest HIV epidemic globally, also predominated by HIV-1C. A total of 44 positions displayed statistically significant differential (p < 0.05) Shannon entropy in the two regions. This comparison also identified 11 codon sites undergoing distinct selection, 8 of which remained differentially selected in an extended comparison of data from Asia (N = 137) and Africa(N = 383). Assessment of correlated mutation networks associated with differentially selected residues revealed common as well as distinct interaction networks. Furthermore, codon usage analysis revealed 17 differentially selected codons (Mann-Whitney test, p < 0.001) in Asia and Africa. Dissimilar trends in GC content across codon positions were also observed. In depth understanding of these divergent evolutionary signatures through extended analysis with larger data-sets would assist development of effective interventions being considered for HIV-1C.

Delineation of Homeostatic Immune Signatures Defining Viremic Non-progression in HIV-1 Infection.

Viremic non-progressors (VNPs), a distinct group of HIV-1-infected individuals, exhibit no signs of disease progression and maintain persistently elevated CD4+ T cell counts for several years despite high viral replication. Comprehensive characterization of homeostatic cellular immune signatures in VNPs can provide unique insights into mechanisms responsible for coping with viral pathogenesis as well as identifying strategies for immune restoration under clinically relevant settings such as antiretroviral therapy (ART) failure. We report a novel homeostatic signature in VNPs, the preservation of the central memory CD4+ T cell (CD4+ T CM ) compartment. In addition, CD4+ TCM preservation was supported by ongoing interleukin-7 (IL-7)-mediated thymic repopulation of naive CD4+ T cells leading to intact CD4+ T cell homeostasis in VNPs. Regulatory T cell (Treg) expansion was found to be a function of preserved CD4+ T cell count and CD4+ T cell activation independent of disease status. However, in light of continual depletion of CD4+ T cell count in progressors but not in VNPs, Tregs appear to be involved in lack of disease progression despite high viremia. In addition to these homeostatic mechanisms resisting CD4+ T cell depletion in VNPs, a relative diminution of terminally differentiated effector subset was observed exclusively in these individuals that might ameliorate consequences of high viral replication. VNPs also shared signatures of impaired CD8+ T cell cytotoxic function with progressors evidenced by increased exhaustion (PD-1 upregulation) and CD127 (IL-7Rα) downregulation contributing to persistent viremia. Thus, the homeostatic immune signatures reported in our study suggest a complex multifactorial mechanism accounting for non-progression in VNPs.

Hepatic interferon γ and tumor necrosis factor a expression in infants with neonatal cholestasis and cytomegalovirus infection.

AIM OF THE STUDY: To determine the hepatic interferon γ (IFN-γ) and tumor necrosis factor α (TNF-α) levels in infants with neonatal cholestasis (NC) and associated cytomegalovirus (CMV) infection. MATERIAL AND METHODS: This study was conducted in 21 infants with NC over a period of 6 months from June 2017 to December 2017 to determine the hepatic IFN-γ and TNF-α levels in infants with NC and associated CMV infection. RESULTS: IFN-γ levels were positive in 16 (80%), low positive in 3 (16%) and negative in 1 (5%) patients. High positive and positive TNF-α levels were seen in 9 (56.3%) patients with positive liver CMV PCR and low positive levels were seen in 7 (43.7%) patients with positive liver CMV PCR (odds ratio [OR] = 2.6). Positive IFN-γ was present in 13 (81.3%) patients with positive liver CMV PCR and low positive or negative IFN-γ was seen in 3 (18.7%) patients with positive liver CMV PCR (OR = 2.2). Six (60%) patients with positive or high positive TNF-α levels in liver tissue had biliary atresia (BA) whereas 7 (77.7%) with low positive TNF-α levels had non-BA neonatal hepatitis (OR = 5.25). Six (37.5%) patients with positive IFN-γ had BA whereas 2 (50%) patients with low positive or negative IFN-γ had BA (OR = 0.6). CONCLUSIONS: There is high prevalence of CMV in liver tissues in patients with NC and elevated TNF-α and IFN-γ levels are seen in these patients. Elevated TNF-α is also seen in patients with BA. The association of elevated TNF-α, BA and CMV infection needs to be evaluated further.

Effect of diversity in gp41 membrane proximal external region of primary HIV-1 Indian subtype C sequences on interaction with broadly neutralizing antibodies 4E10 and 10E8.

Human Immunodeficiency Virus-1 Clade C (HIV-1C) dominates the AIDS epidemic in India, afflicting 2.1 million individuals within the country and more than 15 million people worldwide. Membrane proximal external region (MPER) is an attractive target for broadly neutralizing antibody (bNAb) based therapies. However, information on MPER sequence diversity from India is meagre due to limited sampling of primary viral sequences. In the present study, we examined the variation in MPER of HIV-1C from 24 individuals in Mumbai, India by high throughput sequencing of uncultured viral sequences. Deep sequencing of MPER (662-683; HXB2 envelope amino acid numbering) allowed quantification of intra-individual variation up to 65% at positions 662, 665, 668, 674 and 677 within this region. These variable positions included contact sites targeted by bNAbs 2F5, Z13e1, 4E10 as well as 10E8. Both major and minor epitope variants i.e. 'haplotypes' were generated for each sample dataset. A total of 23, 34 and 25 unique epitope haplotypes could be identified for bNAbs 2F5, Z13e1 and 4E10/10E8 respectively. Further analysis of 4E10 and 10E8 epitopes from our dataset and meta-analysis of previously reported HIV-1 sequences from India revealed 26 epitopes (7 India-specific), heretofore untested for neutralization sensitivity. Peptide-Ab docking predicted 13 of these to be non-binding to 10E8. ELISA, Surface Plasmon Resonance and peptide inhibition of HIV-1 neutralization assays were then performed which validated predicted weak/non-binding interactions for peptides corresponding to six of these epitopes. These results highlight the under-representation of 10E8 non-binding HIV-1C MPER sequences from India. Our study thus underscores the need for increased surveillance of primary circulating envelope sequences for development of efficacious bNAb-based interventions in India.

Detection of Cytomegalovirus in Liver Tissue by Polymerase Chain Reaction in Infants With Neonatal Cholestasis.

AIM: Cytomegalovirus (CMV) is associated with neonatal cholestasis (NC). Diagnosis of CMV infection is most often based on either positive blood CMV IgM or CMV blood polymerase chain reaction (PCR). Isolation of CMV in liver tissues in patients with NC has rarely been reported. This study was undertaken to see if CMV is present in liver tissues of patients with NC and evaluate the correlation between positive CMV PCR in liver tissue with the serology and blood PCR. METHODS: This study was conducted in 31 infants with NC from June 2015 to December 2016. All patients underwent blood CMV IgM, blood CMV PCR and liver CMV PCR. Prevalence of CMV in NC based on positive liver CMV PCR was calculated. Sensitivity and specificity of the serologic markers and blood CMV PCR to identify CMV infection in the liver was determined. RESULTS: CMV IgM was positive in 13 (42%) patients, CMV IgG was positive in 26 (84%) patients and blood CMV PCR was positive in 23 (74%) patients. Liver CMV PCR was positive in 16 (52%) patients. Fifteen (48%) patients had biliary atresia (BA), 10 (32%) patients had neonatal hepatitis, 5 (16%) had paucity of bile ducts and 1 (3%) had ascending cholangitis. Of the 16 patients with positive liver CMV PCR, 8 (50%) had BA, 4 (25%) had neonatal hepatitis, 3 (19%) had paucity of bile ducts and 1 (6%) had ascending cholangitis. Sensitivity of blood CMV IgM in relation to liver CMV PCR was 69% and specificity was 61%. Sensitivity of blood CMV PCR was 61% and specificity was 71% when compared with liver CMV PCR. CONCLUSION: CMV is present in the liver tissues of more than half the patients with NC. Serology or blood CMV PCR is apparently not an accurate marker of CMV in the liver tissue. Also, CMV infection in children seems to be associated equally with BA or non-BA neonatal hepatitis.