Immunotherapeutic approaches to eliminate latently HIV-infected cells are focused on the adaptive immune system. Herein we provide mechanistic evidence for a molecular cascade characterized by epigenetic reprogramming of innate myeloid cells and CD4 T cells. The coordinate regulation and gene expression mediated by transcription factors (TFs) IRF3, IRF7, STAT1 and C/EBPβ versus AP-1, promoted the development of innate antiviral immunity in these cells which was associated with control of viral load and decay of cell associated viral DNA (CA-vDNA) following analytical treatment interruption (ATI) in SIV-infected rhesus macaques (RMs) treated with anti-IL-10 and anti-PD-1. The prevalence of TGF-β/SMAD signaling in a subset of combo-treated RMs with high CA-vDNA (CA-vDNAhi) suppressed this antiviral activity through histone deacetylases, including HDAC11, as the latter reduced chromatin accessibility of IRFs and STATs and impeded their antiviral functions. The addition of HDAC inhibitors in vitro restored antiviral response in the presence of TGF-β. Induction of IL-6, a target gene of C/EBPβ, in CA-vDNAlo RMs, amplified the antiviral network through IRF9, a transcription factor upstream of IRF7. We identified a similar molecular cascade in HIV elite controllers, who maintain low to undetectable viremia and small viral reservoirs without treatment. These data highlight the importance of epigenetic regulation of the host in shaping innate antiviral immune responses that control viral rebound following ATI and reduce the viral reservoir, providing insight into potential strategies for HIV cure interventions.

Nucleoside reverse transcriptase translocation inhibitors (NRTTIs) are potent antiretroviral agents that block HIV replication. A comprehensive lead optimization campaign was undertaken to develop a novel long-acting NRTTI with the potential for extended-duration dosing for HIV prophylaxis. Broad exploration of nucleoside structure-activity relationship (SAR), leveraging ribose core, periphery, and nucleobase modifications, along with systematic progression of compounds of interest through key in vitro and in vivo studies led to the discovery of MK-8527. MK-8527 is a novel deoxyadenosine analog that is phosphorylated intracellularly to its active triphosphate (TP) form, which inhibits reverse transcription. Iron footprinting and primer extension assays demonstrated that MK-8527-TP inhibits translocation of reverse transcriptase on the primer and template, and this inhibition allows for both immediate and delayed chain termination of reverse transcription. MK-8527 inhibits viral replication in human peripheral blood mononuclear cells (PBMCs), with a half maximal inhibitory concentration (IC50) of 0.21 nM. The pharmacokinetic (PK) profile of MK-8527 in rats and rhesus monkeys was characterized by low-to-moderate clearance and volume of distribution, with good oral absorption (57% and 100% in rats and monkeys, respectively). Following oral administration of MK-8527 to monkeys, MK-8527-TP exhibited an intracellular half-life of approximately 48 h in PBMCs, significantly longer than the apparent plasma half-life of the parent compound (approximately 7 h). MK-8527 and MK-8527-TP demonstrated favorable in vitro off-target profiles, with IC50 values of ≥95 µM against human DNA polymerases tested, and no off-target activities at 10 μM against a panel of 114 enzyme and receptor binding assays. Collectively, the potent antiretroviral activity and favorable preclinical PK and off-target profiles make MK-8527 an attractive clinical candidate, and it is currently in clinical trials for once-monthly oral HIV-1 pre-exposure prophylaxis.

Immunotherapeutic approaches to eliminate latently HIV-infected cells are focused on the adaptive immune system. Herein we provide mechanistic evidence for a molecular cascade highlighted by epigenetic reprogramming of innate myeloid cells mediated by specific transcription factors (IRF-3, IRF-7, STAT-1, and C/EBPβ). This cascade promotes the development of innate antiviral immunity that triggers viral load control and viral DNA (vDNA) decay post-ATI in SIV-infected rhesus macaques (RMs) treated with anti-IL-10 and anti-PD-1. The balance between the antiviral signaling driven by IRFs, STATs, and C/EBPβ and the pro-inflammatory response mediated by AP-1 triggered the decay of vDNA levels in lymph nodes. TGF-β/SMAD signaling suppressed this antiviral activity through HDAC11, which blocked the chromatin accessibility of IRFs/STATs and impeded their antiviral functions. HDAC inhibitors restored this antiviral response in the presence of TGF-β. IL-6 induction, a target of C/EBPβ, amplified the antiviral network through IRF9, a TF upstream of IRF7, a feature specific of RMs with lower vDNA levels. HIV elite controllers, who maintain undetectable viremia and small viral reservoirs without treatment showed similar molecular cascade. In summary, our data highlights the importance of epigenetic regulation in shaping innate antiviral immune response that controls viral rebound and reduces the viral reservoir, providing insight into potential strategies for HIV cure interventions. R37AI141258 P01AI178376 UM1AI164561 Viral Immunology (VIR)

INTRODUCTION:Islatravir (ISL) is a nucleoside reverse transcriptase translocation inhibitor (NRTTI) with robust antiretroviral activity. The efficacy of ISL administered for post-exposure prophylaxis (PEP) was evaluated in a simian immunodeficiency virus (SIV) rhesus macaque intravenous (IV) challenge model. METHODS:Twelve rhesus macaques were challenged with SIVmac251 via IV administration. After 24 hours, six animals received ISL 3.9 mg/kg (the minimum effective dose that gives maximal protection) and six animals were untreated controls. In stage 1, treated animals received 4 weekly oral doses of ISL and were monitored for SIV infection for 7 weeks after the last dose. In stage 2, uninfected, treated animals from stage 1 were challenged similarly; 24 hours after challenge, 3 weekly oral doses of ISL 3.9 mg/kg were initiated. The treated animals were monitored for 7 weeks, as in stage 1. Uninfected, treated animals (from stage 2) entered stage 3. In stage 3, the animals were challenged as in stage 2; 24 hours after challenge, 2 weekly oral doses of ISL 3.9 mg/kg were initiated. The treated animals were monitored for 7 weeks, as before. Finally, in stage 4, uninfected, treated animals were challenged using IV administration and 24 hours later were treated with a single oral dose of ISL 3.9 mg/kg and monitored for 7 weeks. Infection was monitored through plasma viral RNA and proviral DNA amplification. Virus-specific antibody responses were measured using a commercial assay. ISL concentrations in plasma and ISL triphosphate (ISL-TP) levels in peripheral blood mononuclear cells were measured longitudinally. RESULTS:All untreated controls were viraemic 7 days after SIVmac251 IV challenge. All six ISL-treated animals were completely protected in stages 1-3 (Fisher exact test p = 0.0022). In stage 4, two of six ISL-treated animals became infected with wild-type SIVmac251: viraemia was observed at days 14 and 49 in the two animals (Fisher exact test p = 0.06). Both animals had unquantifiable ISL-TP on the day viraemia was observed. CONCLUSIONS:Two weekly oral doses of ISL 3.9 mg/kg, administered 24 hours post IV SIV exposure, prevents infection of rhesus macaques. These results support further investigation of a long-acting oral NRTTI for PEP.

Human immunodeficiency virus (HIV) persistence during antiretroviral therapy (ART) is associated with heightened plasma interleukin-10 (IL-10) levels and PD-1 expression. We hypothesized that IL-10 and PD-1 blockade would lead to control of viral rebound following analytical treatment interruption (ATI). Twenty-eight ART-treated, simian immunodeficiency virus (SIV)mac239-infected rhesus macaques (RMs) were treated with anti-IL-10, anti-IL-10 plus anti-PD-1 (combo) or vehicle. ART was interrupted 12 weeks after introduction of immunotherapy. Durable control of viral rebound was observed in nine out of ten combo-treated RMs for >24 weeks post-ATI. Induction of inflammatory cytokines, proliferation of effector CD8(+) T cells in lymph nodes and reduced expression of BCL-2 in CD4(+) T cells pre-ATI predicted control of viral rebound. Twenty-four weeks post-ATI, lower viral load was associated with higher frequencies of memory T cells expressing TCF-1 and of SIV-specific CD4(+) and CD8(+) T cells in blood and lymph nodes of combo-treated RMs. These results map a path to achieve long-lasting control of HIV and/or SIV following discontinuation of ART.

Antiretroviral therapy inhibits HIV-1 replication but is not curative due to establishment of a persistent reservoir after virus integration into the host genome. Reservoir reduction is therefore an important HIV-1 cure strategy. Some HIV-1 nonnucleoside reverse transcriptase inhibitors induce HIV-1 selective cytotoxicity in vitro but require concentrations far exceeding approved dosages. Focusing on this secondary activity, we found bifunctional compounds with HIV-1-infected cell kill potency at clinically achievable concentrations. These targeted activator of cell kill (TACK) molecules bind the reverse transcriptase-p66 domain of monomeric Gag-Pol and act as allosteric modulators to accelerate dimerization, resulting in HIV-1+ cell death through premature intracellular viral protease activation. TACK molecules retain potent antiviral activity and selectively eliminate infected CD4+ T cells isolated from people living with HIV-1, supporting an immune-independent clearance strategy.

Identifying virus-host interactions on the cell surface can improve our understanding of viral entry and pathogenesis. SARS-CoV-2, the causative agent of the COVID-19 disease, uses ACE2 as a receptor to enter cells. Yet the full repertoire of cell surface proteins that contribute to viral entry is unknown. We developed a photocatalyst-based viral-host protein microenvironment mapping platform (ViraMap) to probe the molecular neighborhood of the SARS-CoV-2 spike protein on the human cell surface. Application of ViraMap to ACE2-expressing cells captured ACE2, the established co-receptor NRP1, and several novel cell surface proteins. We systematically analyzed the relevance of these candidate proteins to SARS-CoV-2 entry by knockdown and overexpression approaches in pseudovirus and authentic infection models and identified PTGFRN and EFNB1 as bona fide viral entry factors. Our results highlight additional host targets that participate in SARS-CoV-2 infection and showcase ViraMap as a powerful platform for defining viral interactions on the cell surface.

Background: HIV cure-directed clinical trials using analytical treatment interruptions (ATIs) require participants to adhere to frequent monitoring visits for viral load tests. Novel viral load monitoring strategies are needed to decrease participant burden during ATIs. Objective: To examine acceptability of a novel home-based blood collection device for viral load testing in the context of two ongoing ATI trials in Philadelphia, PA, United States. Methods: From January 2021 to February 2022, participants completed three in-depth interviews via teleconference during their participation in an ATI: (1) within two weeks of enrollment in the device study, (2) approximately four weeks after beginning to use the device, and (3) within two weeks of the end of the ATI when ART was re-initiated. We used conventional content analysis to analyze the data. Results: We recruited 17 participants: 15 were cisgender males, 1 cisgender female, and 1 transgender woman. We observed an overall 87% success rate in drawing blood with the device from home collection and found overall high acceptance of the device. A mean of 91.5 devices per participant were used for home-based blood collection. Most PWH viewed the device as relatively convenient, painless, easy to use, and a simple solution to frequent blood draws. The main challenge encountered was the inability to completely fill up devices with blood in some cases. Most participants reported positive experiences with mailing blood samples and could see themselves using the device on a regular basis outside of ATIs. Conclusions: Our study showed participant valued the novel home-based peripheral blood collection for viral load testing in the context of ATI trials. More research will be necessary to optimize implementation of the device and to assess whether blood collected can reliably measure viral loads in the context of ATI trials.

Therapeutic interventions to eradicate latent HIV and restore immune function in ART-treated infection have yet to show efficacy. We applied an integrated -omic approach to identify a distinct group of individuals with poor CD4 T-cell reconstitution (Senescent Immunologic non-responders, “Senescent-INRs”) and the highest frequencies of CD4 T cells with inducible HIV. Contrary to the notion that immune activation drives HIV persistence and immune dysfunction, leukocytes of these subjects expressed genes that regulate cellular senescence. Here, increased frequencies of Tregs and TGF-β signaling cascade expression coincided with downregulation of cell metabolism/cycling in PD1-expressing central memory CD4 T cells and with frequencies of cells with inducible HIV. This profile, driven by a β-hydroxybutyrate rich metabolic milieu, resulted in TGF-β associated latency establishment/maintenance. Our findings identify cellular senescence as a novel mechanism of HIV persistence, that can be targeted by PD-1 or TGF-β specific interventions that have shown safety and efficacy in cancer.

Interleukin-10 (IL-10) is an immunosuppressive cytokine that signals through STAT3 to regulate T follicular helper (Tfh) cell differentiation and germinal center formation. In SIV-infected macaques, levels of IL-10 in plasma and lymph nodes (LNs) were induced by infection and not normalized with antiretroviral therapy (ART). During chronic infection, plasma IL-10 and transcriptomic signatures of IL-10 signaling were correlated with the cell-associated SIV-DNA content within LN CD4+ memory subsets, including Tfh cells, and predicted the frequency of CD4+ Tfh cells and their cell-associated SIV-DNA content during ART, respectively. In ART-treated rhesus macaques, cells harboring SIV-DNA by DNAscope were preferentially found in the LN B cell follicle in proximity to IL-10. Finally, we demonstrated that the in vivo neutralization of soluble IL-10 in ART-treated, SIV-infected macaques reduced B cell follicle maintenance and, by extension, LN memory CD4+ T cells, including Tfh cells and those expressing PD-1 and CTLA-4. Thus, these data support a role for IL-10 in maintaining a pool of target cells in lymphoid tissue that serve as a niche for viral persistence. Targeting IL-10 signaling to impair CD4+ T cell survival and improve antiviral immune responses may represent a novel approach to limit viral persistence in ART-suppressed people living with HIV.

Dynamic in vitro antibacterial studies provide valuable insight on effective dosing strategies prior to translating to in vivo models. Frequent sampling is required to monitor the pharmacodynamics (PD) of these studies, leading to significant work when quantifying the bacterial load of the samples. Spreading a bacterial suspension on agar to allow colony counting is a proven process for measuring very low levels of growth, but commercial automation equipment to handle agar plating and colony counting at scale is not readily available. We describe a process to greatly decrease the hands-on time required for PD assays by utilizing general-purpose liquid handling robots to plate bacteria and a custom-made plate imager to automate colony counting. The platform developed handles the biological assay from beginning to end as well as sample tracking at each step of the process. The process relies heavily on custom automation scheduling software to enable dynamic process decisions and coordinate data flow throughout. Using the described platform, we can efficiently quantify >100 PD samples per day while maintaining the necessary dynamic range of the assay. Alleviating the main bottleneck in the dynamic antibacterial studies has allowed us to accelerate the rate of experiments to provide antibacterial dosing data within shorter timelines.

Abstract Background Molnupiravir is an orally available prodrug of the antiviral nucleoside analog N-Hydroxycytidine (NHC). In preclinical studies NHC has shown broad-spectrum antiviral activity against multiple RNA viruses including SARS-CoV-2. Incorporation of NHC by viral polymerases impairs replication by introducing errors into the viral genome. NHC has been shown to have a high barrier to the development of resistance in vitro with RSV, Influenza and Venezualen Equine Encephalitis viruses. In these studies, we have explored the potential for SARS-CoV-2 to develop resistance to NHC in cell culture. Methods Vero E6 cells were infected with SARS-CoV-2 (WA-1) in triplicate in the presence of NHC or a C3L-protease inhibitor (MRK-A). Culture supernatants from wells with the highest drug concentration exhibiting a cytopathic effect (CPE) score of ≥ 2+ were repassaged and at each passage, IC50 values were estimated based on CPE scoring. At each passage, full genome next generation sequencing (NGS) was performed on the viral RNA Results No change in susceptibility to NHC (EC50 fold change ≤ 1.1) was noted in 2 of 3 cultures and a 2-fold change was observed in one culture after 30 passages. In contrast, a 3- to 4-fold decreases in susceptibility to the 3CL protease inhibitor were seen by passage by 12, with increasing resistance of 4.6- to 15.7-fold observed by passage 30. NHC passaged viruses exhibited 53 to 99 amino acid changes, including substitutions and deletions (both in-frame and frameshift), across 25 different viral proteins as compared with 10 to 13 changes in 13 proteins in the MRK-A cultures. With NHC, 3 to 4 changes were observed in the viral polymerase; however, these were randomly distributed, and none were observed more than once. In contrast, the 3CL protease passaged virus had a nsp5 T21I substitution detected in all 3 cultures. Conclusion No evidence of SARS-CoV-2 phenotypic or genotypic resistance was observed following 30 passages with NHC. A random pattern of amino acid changes were observed across multiple proteins consistent with the mechanism of action of NHC. In the same study, resistance was readily selected to a control 3CL protease inhibitor. Together these data support previous reports demonstrating the high barrier to resistance of NHC. Disclosures Julie Strizki, PhD, Merck and Co.: Stocks/Bonds Nicholas Murgolo, PhD, MERCK: Employee|MERCK: Stocks/Bonds John Howe, PhD, Merck & Co Inc: Employee|Merck & Co Inc: Stocks/Bonds Beth Hutchins, PhD, Merck & Co.: Employee|Merck & Co.: Stocks/Bonds Hiroshi Mohri, PhD/MD, Merck: Grant/Research Support Daria Hazuda, PhD, Merck: Employee|Merck: Stocks/Bonds Jay Grobler, PhD, Merck & Co., Inc.: Employee|Merck & Co., Inc.: Stocks/Bonds.

Doravirine (DOR), a non-nucleoside reverse transcriptase inhibitor (NNRTI), was approved for treatment of HIV-1 infection in 2018. In the pivotal phase 3 trials, DRIVE-FORWARD and DRIVE-AHEAD, 7 out of 747 (0.9%) treatment-naive participants treated with DOR plus two nucleos(t)ide reverse transcriptase inhibitors (NRTIs) met protocol-defined virologic failure criteria and showed phenotypic resistance to DOR at week 48. The most common DOR resistance-associated mutation (RAM) detected in 5 of the 7 resistant isolates was F227C. Six isolates bearing NRTI RAMs (M184V and/or K65R) were resistant to lamivudine (3TC) and emtricitabine (FTC) but not to other approved NRTIs. All DOR-resistant isolates were susceptible or hypersusceptible (fold change of <0.25) to islatravir (ISL), a nucleoside reverse transcriptase translocation inhibitor (NRTTI). Isolate hypersusceptibility to ISL required F227C, in contrast to zidovudine, an NRTI, which required M184V. Based on the frequent emergence of F227C, we hypothesized that DOR and ISL would create a combination (DOR/ISL) with a high barrier to resistance. In de novo resistance selection studies in MT4-GFP cells (MT4 cells engineered to express green fluorescent protein), DOR/ISL synergistically prevented viral breakthrough at a threshold of 2× the half-maximal inhibitory concentration (IC50). DOR/ISL exhibited a higher barrier to resistance than DOR/3TC and dolutegravir (DTG)/3TC. Resistance analysis showed no emergence of substitutions at F227, an observation consistent with its ability to confer hypersusceptibility to ISL. Overall, the data demonstrate that DOR/ISL creates a 2-drug combination with a higher barrier to resistance, consistent with the reported clinical activity.

Background People with HIV (PWH) and community members have advocated for the development of a home-based viral load test device that could make analytical treatment interruptions (ATIs) less burdensome. Objective We assessed community acceptability of a novel home-based viral load test device. Methods In 2021, we conducted 15 interviews and 3 virtual focus groups with PWH involved in HIV cure research. We used conventional thematic analysis to analyze the data. Results PWH viewed the home-based viral load test device as a critical adjunct in ongoing HIV cure trials with ATIs. The ability to test for viral load at home on demand would alleviate anxiety around being off ART. Participants drew parallels with glucometers used for diabetes. A preference was expressed for the home-based test to clearly indicate whether one was detectable or undetectable for HIV to mitigate risk of HIV transmission to partners. Perceived advantages of the device included convenience, sense of control, and no puncturing of veins. Perceived concerns were possible physical marks, user errors and navigating the logistics of mailing samples to a laboratory and receiving test results. Participants expressed mixed effects on stigma, such as helping normalize HIV, but increased potential for inadvertent disclosure of HIV status or ATI participation. Increasing pluri-potency of the device beyond viral load testing (e.g., CD4+ count test) would increase its utility. Participants suggested pairing the device with telemedicine and mobile health technologies. Conclusions If proven effective, the home-based viral load test device will become a critical adjunct in HIV cure research and HIV care.

ADVERTISEMENT RETURN TO ARTICLES ASAPPREVEditorialNEXTCall for Papers: Drug Resistance in Infectious Diseases and BeyondCelia A. Schiffer*Celia A. Schiffer*Email: [email protected]More by Celia A. Schifferhttps://orcid.org/0000-0003-2270-6613 and Daria J. HazudaDaria J. HazudaMore by Daria J. Hazudahttps://orcid.org/0000-0002-6894-1056Cite this: ACS Infect. Dis. 2022, XXXX, XXX, XXX-XXXPublication Date (Web):March 9, 2022Publication History Received21 February 2022Published online9 March 2022https://doi.org/10.1021/acsinfecdis.2c00103Published 2022 by American Chemical SocietyRIGHTS & PERMISSIONSArticle Views412Altmetric-Citations-LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (346 KB) Get e-Alerts Get e-Alerts

Frequent viral load testing is necessary during analytical treatment interruptions (ATIs) in HIV cure-directed clinical trials, though such may be burdensome and inconvenient to trial participants. We implemented a national, cross-sectional survey in the United States to examine the acceptability of a novel home-based peripheral blood collection device for HIV viral load testing. Between June and August 2021, we distributed an online survey to people with HIV (PWH) and community members, biomedical HIV cure researchers and HIV care providers. We performed descriptive analyses to summarize the results. We received 73 survey responses, with 51 from community members, 12 from biomedical HIV cure researchers and 10 from HIV care providers. Of those, 51 (70%) were cisgender men and 50 (68%) reported living with HIV. Most (>80% overall) indicated that the device would be helpful during ATI trials and they would feel comfortable using it themselves or recommending it to their patients/participants. Of the 50 PWH, 42 (84%) indicated they would use the device if they were participating in an ATI trial and 27 (54%) also expressed a willingness to use the device outside of HIV cure studies. Increasing sensitivity of viral load tests and pluri-potency of the device (CD4 count, chemistries) would augment acceptability. Survey findings provide evidence that viral load home testing would be an important adjunct to ongoing HIV cure-directed trials involving ATIs. Survey findings may help inform successful implementation and uptake of the device in the context of personalized HIV care.

The growing appreciation of immune cell-cell interactions within disease environments has led to extensive efforts to develop immunotherapies. However, characterizing complex cell-cell interfaces in high resolution remains challenging. Thus, technologies leveraging therapeutic-based modalities to profile intercellular environments offer opportunities to study cell-cell interactions with molecular-level insight. We introduce photocatalytic cell tagging (PhoTag) for interrogating cell-cell interactions using single-domain antibodies (VHHs) conjugated to photoactivatable flavin-based cofactors. Following irradiation with visible light, the flavin photocatalyst generates phenoxy radical tags for targeted labeling. Using this technology, we demonstrate selective synaptic labeling across the PD-1/PD-L1 axis in antigen-presenting cell-T cell systems. In combination with multiomics single-cell sequencing, we monitored interactions between peripheral blood mononuclear cells and Raji PD-L1 B cells, revealing differences in transient interactions with specific T cell subtypes. The utility of PhoTag in capturing cell-cell interactions will enable detailed profiling of intercellular communication across different biological systems.

In response to infection or immunization, antibodies are produced that provide protection against re-exposure with the same pathogen. These antibodies can persist at high titers for decades and are maintained by bone marrow-resident long-lived plasma cells (LLPC). However, the durability of antibody responses to immunization varies amongst vaccines. It is unknown what factors contribute to the differential longevity of serum antibody responses and whether heterogeneity in LLPC contributes to this phenomenon. While LLPC differentiation has been studied extensively in mice, little is known about this population in humans or non-human primates (NHP). Here, we use multi-omic single-cell profiling to identify and characterize the LLPC compartment in NHP. We identify LLPC biomarkers including the marker CD102 and show that CD102 in combination with CD31 identifies LLPC in NHP bone marrow. Additionally, we find that CD102 is expressed by LLPC in mouse and humans. These results further our understanding of the LLPC compartment in NHP, identify biomarkers of LLPC, and provide tissue-specific single cell references for future studies.

Follicular helper T (Tfh) cells promote, whereas follicular regulatory T (Tfr) cells restrain, germinal center (GC) reactions. However, the precise roles of these cells in the complex GC reaction remain poorly understood. Here, we perturb Tfh or Tfr cells after SARS-CoV-2 spike protein vaccination in mice. We find that Tfh cells promote the frequency and somatic hypermutation (SHM) of Spike-specific GC B cells and regulate clonal diversity. Tfr cells similarly control SHM and clonal diversity in the GC but do so by limiting clonal competition. In addition, deletion of Tfh or Tfr cells during primary vaccination results in changes in SHM after vaccine boosting. Aged mice, which have altered Tfh and Tfr cells, have lower GC responses, presenting a bimodal distribution of SHM. Together, these data demonstrate that GC responses to SARS-CoV-2 spike protein vaccines require a fine balance of positive and negative follicular T cell help to optimize humoral immunity.