Epidemiological studies suggest that India has the largest number of dengue virus infection cases worldwide. However, there is minimal information about the immunological responses in these patients. CD8 T cells are important in dengue, because they have been implicated in both protection and immunopathology. Here, we provide a detailed analysis of HLA-DR+ CD38+ and HLA-DR− CD38+ effector CD8 T cell subsets in dengue patients from India and Thailand. Both CD8 T cell subsets expanded and expressed markers indicative of antigen-driven proliferation, tissue homing, and cytotoxic effector functions, with the HLA-DR+ CD38+ subset being the most striking in these effector qualities. The breadth of the dengue-specific CD8 T cell response was diverse, with NS3-specific cells being the most dominant. Interestingly, only a small fraction of these activated effector CD8 T cells produced gamma interferon (IFN-γ) when stimulated with dengue virus peptide pools. Transcriptomics revealed downregulation of key molecules involved in T cell receptor (TCR) signaling. Consistent with this, the majority of these CD8 T cells remained IFN-γ unresponsive even after TCR-dependent polyclonal stimulation (anti-CD3 plus anti-CD28) but produced IFN-γ by TCR-independent polyclonal stimulation (phorbol 12-myristate 13-acetate [PMA] plus ionomycin). Thus, the vast majority of these proliferating, highly differentiated effector CD8 T cells probably acquire TCR refractoriness at the time the patient is experiencing febrile illness that leads to IFN-γ unresponsiveness. Our studies open novel avenues for understanding the mechanisms that fine-tune the balance between CD8 T cell-mediated protective versus pathological effects in dengue.IMPORTANCE Dengue is becoming a global public health concern. Although CD8 T cells have been implicated both in protection and in the cytokine-mediated immunopathology of dengue, how the balance is maintained between these opposing functions remains unknown. We comprehensively characterized CD8 T cell subsets in dengue patients from India and Thailand and show that these cells expand massively and express phenotypes indicative of overwhelming antigenic stimulus and tissue homing/cytotoxic-effector functions but that a vast majority of them fail to produce IFN-γ in vitro. Interestingly, the cells were fully capable of producing the cytokine when stimulated in a T cell receptor (TCR)-independent manner but failed to do so in TCR-dependent stimulation. These results, together with transcriptomics, revealed that the vast majority of these CD8 T cells from dengue patients become cytokine unresponsive due to TCR signaling insufficiencies. These observations open novel avenues for understanding the mechanisms that fine-tune the balance between CD8-mediated protective versus pathological effects.

In this study, by characterizing several human monoclonal antibodies (mAbs) isolated from single B cells of the COVID-19–recovered individuals in India who experienced ancestral Wuhan strain (WA.1) of SARS-CoV-2 during early stages of the pandemic, we found a receptor binding domain (RBD)–specific mAb 002-S21F2 that has rare gene usage and potently neutralized live viral isolates of SARS-CoV-2 variants including Alpha, Beta, Gamma, Delta, and Omicron sublineages (BA.1, BA.2, BA.2.12.1, BA.4, and BA.5) with IC 50 ranging from 0.02 to 0.13 μg/ml. Structural studies of 002-S21F2 in complex with spike trimers of Omicron and WA.1 showed that it targets a conformationally conserved epitope on the outer face of RBD (class 3 surface) outside the ACE2-binding motif, thereby providing a mechanistic insights for its broad neutralization activity. The discovery of 002-S21F2 and the broadly neutralizing epitope it targets have timely implications for developing a broad range of therapeutic and vaccine interventions against SARS-CoV-2 variants including Omicron sublineages.

Broadly neutralizing antibodies (bNAbs) have demonstrated protective effects against HIV-1 in primate studies and recent human clinical trials. Elite neutralizers are potential candidates for isolation of HIV-1 bNAbs. The coexistence of bNAbs such as BG18 with neutralization-susceptible autologous viruses in an HIV-1-infected adult elite controller has been suggested to control viremia. Disease progression is faster in HIV-1-infected children than in adults. Plasma bNAbs with multiple epitope specificities are developed in HIV-1 chronically infected children with more potency and breadth than in adults. Therefore, we evaluated the specificity of plasma neutralizing antibodies of an antiretroviral-naive HIV-1 clade C chronically infected pediatric elite neutralizer, AIIMS_330. The plasma antibodies showed broad and potent HIV-1 neutralizing activity with >87% (29/33) breadth, a median inhibitory dilution (ID50) value of 1,246, and presence of N160 and N332 supersite-dependent HIV-1 bNAbs. The sorting of BG505.SOSIP.664.C2 T332N gp140 HIV-1 antigen-specific single B cells of AIIMS_330 resulted in the isolation of an HIV-1 N332 supersite-dependent bNAb, AIIMS-P01. The AIIMS-P01 neutralized 67% of HIV-1 cross-clade viruses, exhibited substantial indels despite limited somatic hypermutations, interacted with native-like HIV-1 trimer as observed in negative stain electron microscopy, and demonstrated high binding affinity. In addition, AIIMS-P01 neutralized the coexisting and evolving autologous viruses, suggesting the coexistence of vulnerable autologous viruses and HIV-1 bNAbs in the AIIMS_330 pediatric elite neutralizer. Such pediatric elite neutralizers can serve as potential candidates for isolation of novel HIV-1 pediatric bNAbs and for understanding the coevolution of virus and host immune response. IMPORTANCE More than 50% of the HIV-1 infections globally are caused by clade C viruses. To date, there is no effective vaccine to prevent HIV-1 infection. Based on the structural information of the currently available HIV-1 bNAbs, attempts are under way to design immunogens that can elicit correlates of protection upon vaccination. Here, we report the isolation and characterization of an HIV-1 N332 supersite-dependent bNAb, AIIMS-P01, from a clade C chronically infected pediatric elite neutralizer. The N332 supersite is an important epitope and is one of the current HIV-1 vaccine targets. AIIMS-P01 potently neutralized the contemporaneous and autologous evolving viruses and exhibited substantial indels despite low somatic hypermutations. Taken together with the information on infant bNAbs, further isolation and characterization of bNAbs contributing to the plasma breadth in HIV-1 chronically infected children may help provide a better understanding of their role in controlling HIV-1 infection.

Keywords 23 HIV-1, clade C, N332 supersite, plasma neutralization, pediatric HIV-1 bNAb, single B cell 24 sorting, elite neutralizer, SOSIP trimer, negative stain EM and 3D reconstruction 25 2 Abstract 26 27 Broadly neutralizing antibodies (bNAbs) have demonstrated protective effects against HIV-1 28 in primate studies and recent human clinical trials. Elite-neutralizers are potential candidates 29 for isolation of HIV-1 bNAbs and coexistence of bNAbs such as BG18 with neutralization 30 susceptible autologous viruses in an HIV-1 infected adult elite controller has been suggested 31 to control viremia. Disease progression is faster in HIV-1 infected children than adults. 32 Plasma bNAbs with multiple epitope specificities are developed in HIV-1 chronically 33 infected children with more potency and breadth than in adults. Therefore, we evaluated the 34 specificity of plasma neutralizing antibodies of an antiretroviral naïve HIV-1 clade C 35 chronically infected pediatric elite neutralizer AIIMS_330. The plasma antibodies showed 36 broad and potent HIV-1 neutralizing activity with >87% (29/33) breadth, median inhibitory 37 dilution (ID50) value of 1246 and presence of N160 and N332-supersite dependent HIV-1 38 bNAbs. The sorting of BG505.SOSIP.664.C2 T332N gp140 HIV-1 antigen-specific single B 39 cells of AIIMS_330 resulted in the isolation of an HIV-1 N332-supersite dependent bNAb 40 AIIMS-P01. The AIIMS-P01 neutralized 67% of HIV-1 cross-clade viruses; exhibited 41 substantial indels despite limited somatic hypermutations; interacted with native-like HIV-1 42 trimer as observed in negative stain electron microscopy and demonstrated high binding 43 affinity. In addition, AIIMS-P01 potently neutralized the coexisting and evolving autologous 44 viruses suggesting the coexistence of vulnerable autologous viruses and HIV-1 bNAbs in 45 AIIMS_330 pediatric elite neutralizer. Further studies on such pediatric elite-neutralizers and 46 isolation of novel HIV-1 pediatric bNAbs may provide newer insights to guide vaccine 47 design.48 49 Total Words: 238 50 51 3 Importance 52 53 More than 50% of the HIV-1 infections globally are caused by clade C viruses. Till date, 54 there is no effective vaccine to prevent HIV-1 infection. Based on the structural information 55 of the currently available HIV-1 bNAbs, attempts are underway to design immunogens that 56 can elicit correlates of protection upon vaccination. Here we report the isolation and 57 characterization of an HIV-1 N332-supersite dependent bNAb AIIMS-P01 from a clade C 58 chronically infected pediatric elite neutralizer. The N332-supersite is an important epitope 59 and is one of the current HIV-1 vaccine targets. AIIMS-P01 potently neutralized the 60 contemporaneous and autologous evolving viruses and exhibits substantial indels despite low 61 somatic hypermutations. Taken together with the information on infant bNAbs, further 62 isolation of bNAbs contributing to the plasma breadth in HIV-1 infected children may help to 63 better understand their development and chara...