The pattern of viral diversification in newly infected individuals provides information about the host environment and immune responses typically experienced by the newly transmitted virus. For example, sites that tend to evolve rapidly across multiple early-infection patients could be involved in enabling escape from common early immune responses, could represent adaptation for rapid growth in a newly infected host, or could represent reversion from less fit forms of the virus that were selected for immune escape in previous hosts. Here we investigated the diversification of HIV-1 env coding sequences in 81 very early B subtype infections previously shown to have resulted from transmission or expansion of single viruses (n = 78) or two closely related viruses (n = 3). In these cases, the sequence of the infecting virus can be estimated accurately, enabling inference of both the direction of substitutions as well as distinction between insertion and deletion events. By integrating information across multiple acutely infected hosts, we find evidence of adaptive evolution of HIV-1 env and identify a subset of codon sites that diversified more rapidly than can be explained by a model of neutral evolution. Of 24 such rapidly diversifying sites, 14 were either i) clustered and embedded in CTL epitopes that were verified experimentally or predicted based on the individual's HLA or ii) in a nucleotide context indicative of APOBEC-mediated G-to-A substitutions, despite having excluded heavily hypermutated sequences prior to the analysis. In several cases, a rapidly evolving site was embedded both in an APOBEC motif and in a CTL epitope, suggesting that APOBEC may facilitate early immune escape. Ten rapidly diversifying sites could not be explained by CTL escape or APOBEC hypermutation, including the most frequently mutated site, in the fusion peptide of gp41. We also examined the distribution, extent, and sequence context of insertions and deletions, and we provide evidence that the length variation seen in hypervariable loop regions of the envelope glycoprotein is a consequence of selection and not of mutational hotspots. Our results provide a detailed view of the process of diversification of HIV-1 following transmission, highlighting the role of CTL escape and hypermutation in shaping viral evolution during the establishment of new infections.

Identifying microbial pathogens with zoonotic potential in wild-living primates can be important to human health, as evidenced by human immunodeficiency viruses types 1 and 2 (HIV-1 and HIV-2) and Ebola virus. Simian foamy viruses (SFVs) are ancient retroviruses that infect Old and New World monkeys and apes. Although not known to cause disease, these viruses are of public health interest because they have the potential to infect humans and thus provide a more general indication of zoonotic exposure risks. Surprisingly, no information exists concerning the prevalence, geographic distribution, and genetic diversity of SFVs in wild-living monkeys and apes. Here, we report the first comprehensive survey of SFVcpz infection in free-ranging chimpanzees (Pan troglodytes) using newly developed, fecal-based assays. Chimpanzee fecal samples (n = 724) were collected at 25 field sites throughout equatorial Africa and tested for SFVcpz-specific antibodies (n = 706) or viral nucleic acids (n = 392). SFVcpz infection was documented at all field sites, with prevalence rates ranging from 44% to 100%. In two habituated communities, adult chimpanzees had significantly higher SFVcpz infection rates than infants and juveniles, indicating predominantly horizontal rather than vertical transmission routes. Some chimpanzees were co-infected with simian immunodeficiency virus (SIVcpz); however, there was no evidence that SFVcpz and SIVcpz were epidemiologically linked. SFVcpz nucleic acids were recovered from 177 fecal samples, all of which contained SFVcpz RNA and not DNA. Phylogenetic analysis of partial gag (616 bp), pol-RT (717 bp), and pol-IN (425 bp) sequences identified a diverse group of viruses, which could be subdivided into four distinct SFVcpz lineages according to their chimpanzee subspecies of origin. Within these lineages, there was evidence of frequent superinfection and viral recombination. One chimpanzee was infected by a foamy virus from a Cercopithecus monkey species, indicating cross-species transmission of SFVs in the wild. These data indicate that SFVcpz (i) is widely distributed among all chimpanzee subspecies; (ii) is shed in fecal samples as viral RNA; (iii) is transmitted predominantly by horizontal routes; (iv) is prone to superinfection and recombination; (v) has co-evolved with its natural host; and (vi) represents a sensitive marker of population structure that may be useful for chimpanzee taxonomy and conservation strategies.

Hepatitis C virus (HCV) infection is characterized by persistent replication of a complex mixture of viruses termed a "quasispecies." Transmission is generally associated with a stringent population bottleneck characterized by infection by limited numbers of "transmitted/founder" (T/F) viruses. Characterization of T/F genomes of human immunodeficiency virus type 1 (HIV-1) has been integral to studies of transmission, immunopathogenesis, and vaccine development. Here, we describe the identification of complete T/F genomes of HCV by single-genome sequencing of plasma viral RNA from acutely infected subjects. A total of 2,739 single-genome-derived amplicons comprising 10,966,507 bp from 18 acute-phase and 11 chronically infected subjects were analyzed. Acute-phase sequences diversified essentially randomly, except for the poly(U/UC) tract, which was subject to polymerase slippage. Fourteen acute-phase subjects were productively infected by more than one genetically distinct virus, permitting assessment of recombination between replicating genomes. No evidence of recombination was found among 1,589 sequences analyzed. Envelope sequences of T/F genomes lacked transmission signatures that could distinguish them from chronic infection viruses. Among chronically infected subjects, higher nucleotide substitution rates were observed in the poly(U/UC) tract than in envelope hypervariable region 1. Fourteen full-length molecular clones with variable poly(U/UC) sequences corresponding to seven genotype 1a, 1b, 3a, and 4a T/F viruses were generated. Like most unadapted HCV clones, T/F genomes did not replicate efficiently in Huh 7.5 cells, indicating that additional cellular factors or viral adaptations are necessary for in vitro replication. Full-length T/F HCV genomes and their progeny provide unique insights into virus transmission, virus evolution, and virus-host interactions associated with immunopathogenesis.

Both the recognition of HIV-infected cells and the immunogenicity of candidate CTL vaccines depend on the presentation of a peptide epitope at the cell surface, which in turn depends on intracellular antigen processing. Differential antigen processing maybe responsible for the differences in both the quality and the quantity of epitopes produced, influencing the immunodominance hierarchy of viral epitopes. Previously, we showed that the magnitude of the HIV-2 gag-specific T-cell response is inversely correlated with plasma viral load, particularly when responses are directed against an epitope, 165 DRFYKSLRA173 , within the highly conserved Major Homology Region of gag-p26. We also showed that the presence of three proline residues, at positions 119, 159 and 178 of gag-p26, was significantly correlated with low viral load. Since this proline motif was also associated with stronger gag-specific CTL responses, we investigated the impact of these prolines on proteasomal processing of the protective 165 DRFYKSLRA173 epitope. Our data demonstrate that the 165 DRFYKSLRA173 epitope is most efficiently processed from precursors that contain two flanking proline residues, found naturally in low viral-load patients. Superior antigen processing and enhanced presentation may account for the link between infection with HIV-2 encoding the "PPP-gag" sequence and both strong gag-specific CTL responses as well as lower viral load.

Major histocompatibility complex (MHC) class I molecules determine immune responses to viral infections. These polymorphic cell-surface glycoproteins bind peptide antigens, forming ligands for cytotoxic T and natural killer cell receptors. Under pressure from rapidly evolving viruses, hominoid MHC class I molecules also evolve rapidly, becoming diverse and species-specific. Little is known of the impact of infectious disease epidemics on MHC class I variant distributions in human populations, a context in which the chimpanzee is the superior animal model. Population dynamics of the chimpanzees inhabiting Gombe National Park, Tanzania have been studied for over 50 years. This population is infected with SIVcpz, the precursor of human HIV-1. Because HLA-B is the most polymorphic human MHC class I molecule and correlates strongly with HIV-1 progression, we determined sequences for its ortholog, Patr-B, in 125 Gombe chimpanzees. Eleven Patr-B variants were defined, as were their frequencies in Gombe's three communities, changes in frequency with time, and effect of SIVcpz infection. The growing populations of the northern and central communities, where SIVcpz is less prevalent, have stable distributions comprising a majority of low-frequency Patr-B variants and a few high-frequency variants. Driving the latter to high frequency has been the fecundity of immigrants to the northern community, whereas in the central community, it has been the fecundity of socially dominant individuals. In the declining population of the southern community, where greater SIVcpz prevalence is associated with mortality and emigration, Patr-B variant distributions have been changing. Enriched in this community are Patr-B variants that engage with natural killer cell receptors. Elevated among SIVcpz-infected chimpanzees, the Patr-B*06:03 variant has striking structural and functional similarities to HLA-B*57, the human allotype most strongly associated with delayed HIV-1 progression. Like HLA-B*57, Patr-B*06:03 correlates with reduced viral load, as assessed by detection of SIVcpz RNA in feces.

Broadly cross-reactive neutralizing antibodies (bNabs) represent powerful tools to combat human immunodeficiency virus type 1 (HIV-1) infection. Here, we examined whether HIV-1-specific bNabs are capable of cross-neutralizing distantly related simian immunodeficiency viruses (SIVs) infecting central (Pan troglodytes troglodytes) (SIVcpzPtt) and eastern (Pan troglodytes schweinfurthii) (SIVcpzPts) chimpanzees (n = 11) as well as western gorillas (Gorilla gorilla gorilla) (SIVgor) (n = 1). We found that bNabs directed against the CD4 binding site (n = 10), peptidoglycans at the base of variable loop 3 (V3) (n = 5), and epitopes at the interface of surface (gp120) and membrane-bound (gp41) envelope glycoproteins (n = 5) failed to neutralize SIVcpz and SIVgor strains. In addition, apex V2-directed bNabs (n = 3) as well as llama-derived (heavy chain only) antibodies (n = 6) recognizing both the CD4 binding site and gp41 epitopes were either completely inactive or neutralized only a fraction of SIVcpzPtt strains. In contrast, one antibody targeting the membrane-proximal external region (MPER) of gp41 (10E8), functional CD4 and CCR5 receptor mimetics (eCD4-Ig, eCD4-Ig(mim2), CD4-218.3-E51, and CD4-218.3-E51-mim2), as well as mono- and bispecific anti-human CD4 (iMab and LM52) and CCR5 (PRO140, PRO140-10E8) receptor antibodies neutralized >90% of SIVcpz and SIVgor strains with low-nanomolar (0.13 to 8.4 nM) potency. Importantly, the latter antibodies blocked virus entry not only in TZM-bl cells but also in Cf2Th cells expressing chimpanzee CD4 and CCR5 and neutralized SIVcpz in chimpanzee CD4(+) T cells, with 50% inhibitory concentrations (IC50s) ranging from 3.6 to 40.5 nM. These findings provide new insight into the protective capacity of anti-HIV-1 bNabs and identify candidates for further development to combat SIVcpz infection.

NF-κB is essential for effective transcription of primate lentiviral genomes and also activates antiviral host genes. Here, we show that the early protein Nef of most primate lentiviruses enhances NF-κB activation. In contrast, the late protein Vpu of HIV-1 and its simian precursors inhibits activation of NF-κB, even in the presence of Nef. Although this effect of Vpu did not correlate with its ability to interact with β-TrCP, it involved the stabilization of IκB and reduced nuclear translocation of p65. Interestingly, however, Vpu did not affect casein kinase II-mediated phosphorylation of p65. Lack of Vpu was associated with increased NF-κB activation and induction of interferon and interferon-stimulated genes (ISGs) in HIV-1-infected T cells. Thus, HIV-1 and its simian precursors employ Nef to boost NF-κB activation early during the viral life cycle to initiate proviral transcription, while Vpu is used to downmodulate NF-κB-dependent expression of ISGs at later stages.

African apes harbour at least six Plasmodium species of the subgenus Laverania, one of which gave rise to human Plasmodium falciparum. Here we use a selective amplification strategy to sequence the genome of chimpanzee parasites classified as Plasmodium reichenowi and Plasmodium gaboni based on the subgenomic fragments. Genome-wide analyses show that these parasites indeed represent distinct species, with no evidence of cross-species mating. Both P. reichenowi and P. gaboni are 10-fold more diverse than P. falciparum, indicating a very recent origin of the human parasite. We also find a remarkable Laverania-specific expansion of a multigene family involved in erythrocyte remodelling, and show that a short region on chromosome 4, which encodes two essential invasion genes, was horizontally transferred into a recent P. falciparum ancestor. Our results validate the selective amplification strategy for characterizing cryptic pathogen species, and reveal evolutionary events that likely predisposed the precursor of P. falciparum to colonize humans.

Sexual transmission of human immunodeficiency virus type 1 (HIV-1) most often results from productive infection by a single transmitted/founder (T/F) virus, indicating a stringent mucosal bottleneck. Understanding the viral traits that overcome this bottleneck could have important implications for HIV-1 vaccine design and other prevention strategies. Most T/F viruses use CCR5 to infect target cells and some encode envelope glycoproteins (Envs) that contain fewer potential N-linked glycosylation sites and shorter V1/V2 variable loops than Envs from chronic viruses. Moreover, it has been reported that the gp120 subunits of certain transmitted Envs bind to the gut-homing integrin α4β7, possibly enhancing virus entry and cell-to-cell spread. Here we sought to determine whether subtype C T/F viruses, which are responsible for the majority of new HIV-1 infections worldwide, share biological properties that increase their transmission fitness, including preferential α4β7 engagement. Using single genome amplification, we generated panels of both T/F (n = 20) and chronic (n = 20) Env constructs as well as full-length T/F (n = 6) and chronic (n = 4) infectious molecular clones (IMCs). We found that T/F and chronic control Envs were indistinguishable in the efficiency with which they used CD4 and CCR5. Both groups of Envs also exhibited the same CD4+ T cell subset tropism and showed similar sensitivity to neutralization by CD4 binding site (CD4bs) antibodies. Finally, saturating concentrations of anti-α4β7 antibodies failed to inhibit infection and replication of T/F as well as chronic control viruses, although the growth of the tissue culture-adapted strain SF162 was modestly impaired. These results indicate that the population bottleneck associated with mucosal HIV-1 acquisition is not due to the selection of T/F viruses that use α4β7, CD4 or CCR5 more efficiently.

Single genome sequencing of early HIV-1 genomes provides a sensitive, dynamic assessment of virus evolution and insight into the earliest anti-viral immune responses in vivo. By using this approach, together with deep sequencing, site-directed mutagenesis, antibody adsorptions and virus-entry assays, we found evidence in three subjects of neutralizing antibody (Nab) responses as early as 2 weeks post-seroconversion, with Nab titers as low as 1∶20 to 1∶50 (IC(50)) selecting for virus escape. In each of the subjects, Nabs targeted different regions of the HIV-1 envelope (Env) in a strain-specific, conformationally sensitive manner. In subject CH40, virus escape was first mediated by mutations in the V1 region of the Env, followed by V3. HIV-1 specific monoclonal antibodies from this subject mapped to an immunodominant region at the base of V3 and exhibited neutralizing patterns indistinguishable from polyclonal antibody responses, indicating V1-V3 interactions within the Env trimer. In subject CH77, escape mutations mapped to the V2 region of Env, several of which selected for alterations of glycosylation. And in subject CH58, escape mutations mapped to the Env outer domain. In all three subjects, initial Nab recognition was followed by sequential rounds of virus escape and Nab elicitation, with Nab escape variants exhibiting variable costs to replication fitness. Although delayed in comparison with autologous CD8 T-cell responses, our findings show that Nabs appear earlier in HIV-1 infection than previously recognized, target diverse sites on HIV-1 Env, and impede virus replication at surprisingly low titers. The unexpected in vivo sensitivity of early transmitted/founder virus to Nabs raises the possibility that similarly low concentrations of vaccine-induced Nabs could impair virus acquisition in natural HIV-1 transmission, where the risk of infection is low and the number of viruses responsible for transmission and productive clinical infection is typically one.

Antigens encoded by the var gene family are major virulence factors of the human malaria parasite Plasmodium falciparum, exhibiting enormous intra- and interstrain diversity. Here we use network analysis to show that var architecture and mosaicism are conserved at multiple levels across the Laverania subgenus, based on var-like sequences from eight single-species and three multi-species Plasmodium infections of wild-living or sanctuary African apes. Using select whole-genome amplification, we also find evidence of multi-domain var structure and synteny in Plasmodium gaboni, one of the ape Laverania species most distantly related to P. falciparum, as well as a new class of Duffy-binding-like domains. These findings indicate that the modular genetic architecture and sequence diversity underlying var-mediated host-parasite interactions evolved before the radiation of the Laverania subgenus, long before the emergence of P. falciparum.

Pandemic strains of HIV-1 (group M) encode a total of nine structural (gag, pol, env), regulatory (rev, tat) and accessory (vif, vpr, vpu, nef) genes. However, some subtype A and C viruses exhibit an unusual gene arrangement in which the first exon of rev (rev1) and the vpu gene are placed in the same open reading frame. Although this rev1-vpu gene fusion is present in a considerable fraction of HIV-1 strains, its functional significance is unknown.

Despite the recent development of highly effective anti-hepatitis C virus (HCV) drugs, the global burden of this pathogen remains immense. Control or eradication of HCV will likely require the broad application of antiviral drugs and development of an effective vaccine. A precise molecular identification of transmitted/founder (T/F) HCV genomes that lead to productive clinical infection could play a critical role in vaccine research, as it has for HIV-1. However, the replication schema of these two RNA viruses differ substantially, as do viral responses to innate and adaptive host defenses. These differences raise questions as to the certainty of T/F HCV genome inferences, particularly in cases where multiple closely related sequence lineages have been observed. To clarify these issues and distinguish between competing models of early HCV diversification, we examined seven cases of acute HCV infection in humans and chimpanzees, including three examples of virus transmission between linked donors and recipients. Using single-genome sequencing (SGS) of plasma vRNA, we found that inferred T/F sequences in recipients were identical to viral sequences in their respective donors. Early in infection, HCV genomes generally evolved according to a simple model of random evolution where the coalescent corresponded to the T/F sequence. Closely related sequence lineages could be explained by high multiplicity infection from a donor whose viral sequences had undergone a pretransmission bottleneck due to treatment, immune selection, or recent infection. These findings validate SGS, together with mathematical modeling and phylogenetic analysis, as a novel strategy to infer T/F HCV genome sequences.

Many pathogens evade cytotoxic T lymphocytes (CTLs) by downregulating HLA molecules on infected cells, but the loss of HLA can trigger NK cell-mediated lysis. HIV-1 is thought to subvert CTLs while preserving NK cell inhibition by Nef-mediated downregulation of HLA-A and -B but not HLA-C molecules. We find that HLA-C is downregulated by most primary HIV-1 clones, including transmitted founder viruses, in contrast to the laboratory-adapted NL4-3 virus. HLA-C reduction is mediated by viral Vpu and reduces the ability of HLA-C restricted CTLs to suppress viral replication in CD4+ cells in vitro. HLA-A/B are unaffected by Vpu, and primary HIV-1 clones vary in their ability to downregulate HLA-C, possibly in response to whether CTLs or NK cells dominate immune pressure through HLA-C. HIV-2 also suppresses HLA-C expression through distinct mechanisms, underscoring the immune pressure HLA-C exerts on HIV. This viral immune evasion casts new light on the roles of CTLs and NK cells in immune responses against HIV.

Plasmodium falciparum, the major cause of malaria morbidity and mortality worldwide, is only distantly related to other human malaria parasites and has thus been placed in a separate subgenus, termed Laverania Parasites morphologically similar to P. falciparum have been identified in African apes, but only one other Laverania species, Plasmodium reichenowi from chimpanzees, has been formally described. Although recent studies have pointed to the existence of additional Laverania species, their precise number and host associations remain uncertain, primarily because of limited sampling and a paucity of parasite sequences other than from mitochondrial DNA. To address this, we used limiting dilution polymerase chain reaction to amplify additional parasite sequences from a large number of chimpanzee and gorilla blood and fecal samples collected at two sanctuaries and 30 field sites across equatorial Africa. Phylogenetic analyses of more than 2,000 new sequences derived from the mitochondrial, nuclear, and apicoplast genomes revealed six divergent and well-supported clades within the Laverania parasite group. Although two of these clades exhibited deep subdivisions in phylogenies estimated from organelle gene sequences, these sublineages were geographically defined and not present in trees from four unlinked nuclear loci. This greatly expanded sequence data set thus confirms six, and not seven or more, ape Laverania species, of which P. reichenowi, Plasmodium gaboni, and Plasmodium billcollinsi only infect chimpanzees, whereas Plasmodium praefalciparum, Plasmodium adleri, and Pladmodium blacklocki only infect gorillas. The new sequence data also confirm the P. praefalciparum origin of human P. falciparum.

Mucosal transmission of the human immunodeficiency virus (HIV) results in a bottleneck in viral genetic diversity. Gnanakaran and colleagues used a computational strategy to identify signature amino acids at particular positions in Envelope that were associated either with transmitted sequences sampled very early in infection, or sequences sampled during chronic infection. Among the strongest signatures observed was an enrichment for the stable presence of histidine at position 12 at transmission and in early infection, and a recurrent loss of histidine at position 12 in chronic infection. This amino acid lies within the leader peptide of Envelope, a region of the protein that has been shown to influence envelope glycoprotein expression and virion infectivity. We show a strong association between a positively charged amino acid like histidine at position 12 in transmitted/founder viruses with more efficient trafficking of the nascent envelope polypeptide to the endoplasmic reticulum and higher steady-state glycoprotein expression compared to viruses that have a non-basic position 12 residue, a substitution that was enriched among viruses sampled from chronically infected individuals. When expressed in the context of other viral proteins, transmitted envelopes with a basic amino acid position 12 were incorporated at higher density into the virus and exhibited higher infectious titers than did non-signature envelopes. These results support the potential utility of using a computational approach to examine large viral sequence data sets for functional signatures and indicate the importance of Envelope expression levels for efficient HIV transmission.

Autophagy is a conserved constitutive cellular process, responsible for the degradation of dysfunctional proteins and organelles. Autophagy plays a role in many diseases such as neurodegeneration and cancer; however, to date, conventional autophagy detection techniques are not suitable for clinical samples. We have developed a high throughput, statistically robust technique that quantitates autophagy in primary human leukocytes using the Image stream, an imaging flow cytometer. We validate this method on cell lines and primary cells knocked down for essential autophagy genes. Also, using this method we show that T cells have higher autophagic activity than B cells. Furthermore our results indicate that healthy primary senescent CD8(+) T cells have decreased autophagic levels correlating with increased DNA damage, which may explain features of the senescent immune system and its declining function with age. This technique will allow us, for the first time, to measure autophagy levels in diseases with a known link to autophagy, while also determining the contribution of autophagy to the efficacy of drugs.

CD8+ cytotoxic T lymphocytes (CTLs) play an important role in containment of virus replication in primary human immunodeficiency virus (HIV) infection. HIV's ability to mutate to escape from CTL pressure is increasingly recognized; but comprehensive studies of escape from the CD8 T cell response in primary HIV infection are currently lacking. Here, we have fully characterized the primary CTL response to autologous virus Env, Gag, and Tat proteins in three patients, and investigated the extent, kinetics, and mechanisms of viral escape from epitope-specific components of the response. In all three individuals, we observed variation beginning within weeks of infection at epitope-containing sites in the viral quasispecies, which conferred escape by mechanisms including altered peptide presentation/recognition and altered antigen processing. The number of epitope-containing regions exhibiting evidence of early CTL escape ranged from 1 out of 21 in a subject who controlled viral replication effectively to 5 out of 7 in a subject who did not. Evaluation of the extent and kinetics of HIV-1 escape from >40 different epitope-specific CD8 T cell responses enabled analysis of factors determining escape and suggested that escape is restricted by costs to intrinsic viral fitness and by broad, codominant distribution of CTL-mediated pressure on viral replication.

Interactions between commensal microbes and the immune system are tightly regulated and maintain intestinal homeostasis, but little is known about these interactions in humans. We investigated responses of human CD4+ T cells to the intestinal microbiota. We measured the abundance of T cells in circulation and intestinal tissues that respond to intestinal microbes and determined their clonal diversity. We also assessed their functional phenotypes and effects on intestinal resident cell populations, and studied alterations in microbe-reactive T cells in patients with chronic intestinal inflammation.

T-cell immunity is likely to play a role in protection against SARS-CoV-2 by helping generate neutralizing antibodies. We longitudinally studied CD4 T-cell responses to the M, N, and S structural proteins of SARS-CoV-2 in 21 convalescent individuals. Within the first two months following symptom onset, a majority of individuals (81%) mount at least one CD4 T-cell response, and 48% of individuals mount detectable SARS-CoV-2-specific peripheral T follicular helper cells (pTfh, defined as CXCR5+PD1+ CD4 T cells). SARS-CoV-2-specific pTfh responses across all three protein specificities correlate with antibody neutralization with the strongest correlation observed for S protein-specific responses. When examined over time, pTfh responses increase in frequency and magnitude in convalescence, and robust responses with magnitudes greater than 5% were detected only at the second convalescent visit, an average of 38 days post-symptom onset. These data deepen our understanding of antigen-specific pTfh responses in SARS-CoV-2 infection, suggesting that M and N protein-specific pTfh may also assist in the development of neutralizing antibodies and that pTfh response formation may be delayed in SARS-CoV-2 infection.