Single-cell profiling of lineage determining transcription factors in antigen-specific CD4+ T cells reveals unexpected complexity in recall responses during immune reconstitution.

Recent studies of protein and gene expression at the single-cell level have revealed that the memory T-cell compartment is more heterogeneous than previously acknowledged. Identifying different T helper subsets involved in memory responses at the single-cell level is thus necessary to understand the level of heterogeneity within this population. Antigen-specific CD4+ T cells were measured using the CD25/OX40 assay together with a qualitative multiplex single-cell RT-PCR assay. Transcription profiles and subset proportions within the antigen-specific CD4+ T-cell population were dissected. Cytomegalovirus (CMV)-specific CD4+ T-cell responses skewed toward a Th1 response, whereas Tetanus toxoid responses skewed toward a Th2 type response. Fluctuations in CD4+ T-cell subsets were observed within the HIV-Gag-specific response during ongoing antiretroviral therapy. Strong effector responses (Th1) were observed in early treatment, however with ongoing therapy this effector response significantly decreased in combination with an increase in Tregs and circulating Tfh-like BCL-6+ memory cells. The apparent increase in Tcm in peripheral blood after a several weeks of antiretroviral therapy may be due to Tfh-like cell egress from germinal centers into the periphery.

Persistent survival of prevalent clonotypes within an immunodominant HIV gag-specific CD8+ T cell response.

CD8(+) T cells play a significant role in the control of HIV replication, yet the associated qualitative and quantitative factors that determine the outcome of infection remain obscure. In this study, we examined Ag-specific CD8(+) TCR repertoires longitudinally in a cohort of HLA-B*2705(+) long-term nonprogressors with chronic HIV-1 infection using a combination of molecular clonotype analysis and polychromatic flow cytometry. In each case, CD8(+) T cell populations specific for the immunodominant p24 Gag epitope KRWIILGLNK (KK10; residues 263-272) and naturally occurring variants thereof, restricted by HLA-B*2705, were studied at multiple time points; in addition, comparative data were collected for CD8(+) T cell populations specific for the CMV pp65 epitope NLVPMVATV (NV9; residues 495-503), restricted by HLA-A*0201. Dominant KK10-specific clonotypes persisted for several years and exhibited greater stability than their contemporaneous NV9-specific counterparts. Furthermore, these dominant KK10-specific clonotypes exhibited cross-reactivity with antigenic variants and expressed significantly higher levels of CD127 (IL-7Rα) and Bcl-2. Of note, we also found evidence that promiscuous TCR α-chain pairing associated with alterations in fine specificity for KK10 variants could contribute to TCR ß-chain prevalence. Taken together, these data suggest that an antiapoptotic phenotype and the ability to cross-recognize variant epitopes contribute to clonotype longevity and selection within the peripheral memory T cell pool in the presence of persistent infection with a genetically unstable virus.

SARS-CoV-2 Omicron BA.5: Evolving tropism and evasion of potent humoral responses and resistance to clinical immunotherapeutics relative to viral variants of concern.

BACKGROUND: Genetically distinct viral variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been recorded since January 2020. The introduction of global vaccine programs has contributed to lower COVID-19 hospitalisation and mortality rates, particularly in developed countries. In late 2021, Omicron BA.1 emerged, with substantially altered genetic differences and clinical effects from other variants of concern. Shortly after dominating global spread in early 2022, BA.1 was supplanted by the genetically distinct Omicron lineage BA.2. A sub-lineage of BA.2, designated BA.5, presently has an outgrowth advantage over BA.2 and other BA.2 sub-lineages. Here we study the neutralisation of Omicron BA.1, BA.2 and BA.5 and pre-Omicron variants using a range of vaccine and convalescent sera and therapeutic monoclonal antibodies using a live virus neutralisation assay. Using primary nasopharyngeal swabs, we also tested the relative fitness of BA.5 compared to pre-Omicron and Omicron viral lineages in their ability to use the ACE2-TMPRSS2 pathway. METHODS: Using low passage clinical isolates of Clade A.2.2, Beta, Delta, BA.1, BA.2 and BA.5, we determined humoral neutralisation in vitro in vaccinated and convalescent cohorts, using concentrated human IgG pooled from thousands of plasma donors, and licensed monoclonal antibody therapies. We then determined infectivity to particle ratios in primary nasopharyngeal samples and expanded low passage isolates in a genetically engineered ACE2/TMPRSS2 cell line in the presence and absence of the TMPRSS2 inhibitor Nafamostat. FINDINGS: Peak responses to 3 doses of BNT162b2 vaccine were associated with a 9-fold reduction in neutralisation for Omicron lineages BA.1, BA.2 and BA.5. Concentrated pooled human IgG from convalescent and vaccinated donors and BNT162b2 vaccination with BA.1 breakthrough infections were associated with greater breadth of neutralisation, although the potency was still reduced 7-fold across all Omicron lineages. Testing of clinical grade antibodies revealed a 14.3-fold reduction using Evusheld and 16.8-fold reduction using Sotrovimab for the BA.5. Whilst the infectivity of BA.1 and BA.2 was attenuated in ACE2/TMPRSS2 entry, BA.5 was observed to be equivalent to that of an early 2020 circulating clade and had greater sensitivity to the TMPRSS2 inhibitor Nafamostat. INTERPRETATION: Observations support all Omicron variants to significantly escape neutralising antibodies across a range of vaccination and/or convalescent responses. Potency of therapeutic monoclonal antibodies is also reduced and differs across Omicron lineages. The key difference of BA.5 from other Omicron sub-variants is the reversion in tropism back to using the well-known ACE2-TMPRSS2 pathway, utilised efficiently by pre-Omicron lineages. Monitoring if these changes influence transmission and/or disease severity will be key for ongoing tracking and management of Omicron waves globally. FUNDING: This work was primarily supported by Australian Medical Foundation research grants MRF2005760 (ST, GM & WDR), MRF2001684 (ADK and ST) and Medical Research Future Fund Antiviral Development Call grant (WDR), Medical Research Future Fund COVID-19 grant (MRFF2001684, ADK & SGT) and the New South Wales Health COVID-19 Research Grants Round 2 (SGT).

Platform for isolation and characterization of SARS-CoV-2 variants enables rapid characterization of Omicron in Australia.

Genetically distinct variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have emerged since the start of the COVID-19 pandemic. Over this period, we developed a rapid platform (R-20) for viral isolation and characterization using primary remnant diagnostic swabs. This, combined with quarantine testing and genomics surveillance, enabled the rapid isolation and characterization of all major SARS-CoV-2 variants circulating in Australia in 2021. Our platform facilitated viral variant isolation, rapid resolution of variant fitness using nasopharyngeal swabs and ranking of evasion of neutralizing antibodies. In late 2021, variant of concern Omicron (B1.1.529) emerged. Using our platform, we detected and characterized SARS-CoV-2 VOC Omicron. We show that Omicron effectively evades neutralization antibodies and has a different entry route that is TMPRSS2-independent. Our low-cost platform is available to all and can detect all variants of SARS-CoV-2 studied so far, with the main limitation being that our platform still requires appropriate biocontainment.

High levels of human antigen-specific CD4+ T cells in peripheral blood revealed by stimulated coexpression of CD25 and CD134 (OX40).

Ag-specific human CD4(+) memory T lymphocytes have mostly been studied using assays of proliferation in vitro. Intracellular cytokine and ELISPOT assays quantify effector cell populations but barely detect responses to certain recall Ags that elicit strong proliferative responses, e.g., tetanus toxoid, that comprise non-Th1 CD4(+) cells. We have found that culturing whole blood with Ag for 40-48 h induces specific CD4(+) T cells to simultaneously express CD25 and CD134. This new technique readily detects responses to well-described CD4(+) T cell recall Ags, including preparations of mycobacteria, CMV, HSV-1, influenza, tetanus toxoid, Candida albicans, and streptokinase, as well as HIV-1 peptides, with high specificity. The assay detects much higher levels of Ag-specific cells than intracellular cytokine assays, plus the cells retain viability and can be sorted for in vitro expansion. Furthermore, current in vitro assays for human CD4(+) memory T lymphocytes are too labor-intensive and difficult to standardize for routine diagnostic laboratories, whereas the whole-blood CD25(+)CD134(+) assay combines simplicity of setup with a straightforward cell surface flow cytometry readout. In addition to revealing the true extent of Ag-specific human CD4(+) memory T lymphocytes, its greatest use will be as a simple in vitro monitor of CD4(+) T cell responses to Ags such as tuberculosis infection or vaccines.

IL-7 receptor is expressed on adult pre-B-cell acute lymphoblastic leukemia and other B-cell derived neoplasms and correlates with expression of proliferation and survival markers.

The interleukin (IL)-7 receptor (IL-7R) is expressed on human pre-B but not mature B-cells. We hypothesised that aberrant expression of IL-7R contributes to B-cell oncogenesis. Surface expression of IL-7R components CD127 and CD132, and intracellular Ki-67 and Bcl-2 were examined by flow cytometry on peripheral blood or bone marrow mononuclear cells (PBMC; BMMC) from patients with B-cell derived neoplasms, chronic human immunodeficiency virus type-1 (HIV-1) infection alone, or healthy volunteers. Plasma IL-7, IL-2, IL-4, IL-6, IL-10, IL-21, TNF-alpha, IFN-gamma and BAFF were measured by enzyme-linked immuno-sorbent assay or bead array. The effects of exogenous IL-7 on PBMC and BMMC were examined. CD127 expression was elevated in pre-B-cell acute lymphoblastic leukemia (pre-B-ALL) and in some cases of Non-Hodgkin's Lymphoma (B-NHL). Plasma IL-7 levels were higher in pre-B-ALL, B-cell chronic lymphocytic leukemia (B-CLL) and HIV-1 associated B-NHL (HIV-B-NHL) compared with control groups. CD127+ pre-B-ALL cells had higher expression of Ki-67, Bcl-2 and CD132 than CD127- counterparts. Unlike T-lineage cells, CD127+ pre-B-ALL cells did not down-regulate CD127 in response to exogenous IL-7. Patients with B-cell derived neoplasms had elevated circulating IL-10 and decreased BAFF. These findings support a role for the IL-7/IL-7R system in B-cell oncogenesis.

Immunogenicity assay validation for an HIV vaccine trial: high IFNγ+/IL-2+ CD8+ T cells background in healthy Thais.

In a vaccine trial, assays for vaccine immunogenicity if performed locally will strengthen local site, can save costs and avoid hurdles associated with specimen transport. Here we report the optimization and validation of an Intracellular Cytokine Staining (ICS) assay which was undertaken in preparation for a phase I HIV vaccine trial conducted in Thailand. Intra-, and inter-operator variability were easily established. However, while attempting to set population cut offs for a positive response we found 4/36 (11%) high background responses of IFNγ(+) and/or IL-2(+) CD8(+) T cells (>1%) in normal healthy volunteers. The determinates of these unexpected responses were explored and minimized.

CD127+CCR5+CD38+++ CD4+ Th1 effector cells are an early component of the primary immune response to vaccinia virus and precede development of interleukin-2+ memory CD4+ T cells.

The stages of development of human antigen-specific CD4+ T cells responding to viral infection and their differentiation into long-term memory cells are not well understood. The inoculation of healthy adults with vaccinia virus presents an opportunity to study these events intensively. Between days 11 and 14 postinoculation, there was a peak of proliferating CCR5+CD38+++ CD4+ effector cells which contained the cytotoxic granule marker T-cell intracellular antigen 1 and included gamma interferon (IFN-gamma)-producing vaccinia virus-specific CD4+ T cells. The majority of these initial vaccinia virus-specific CD4+ T cells were CD127+ and produced interleukin-2 (IL-2) but not CTLA-4 in response to restimulation in vitro. Between days 14 and 21, there was a switch from IFN-gamma and IL-2 coexpression to IL-2 production only, coinciding with a resting phenotype and an increased in vitro proliferation response. The early CCR5+CD38+++ vaccinia virus-specific CD4+ T cells were similar to our previous observations of human immunodeficiency virus (HIV)-specific CD4+ T cells in primary HIV type 1 (HIV-1) infection, but the vaccinia virus-specific cells expressed much more CD127 and IL-2 than we previously found in their HIV-specific counterparts. The current study provides important information on the differentiation of IL-2+ vaccinia virus-specific memory cells, allowing further study of antiviral effector CD4+ T cells in healthy adults and their dysfunction in HIV-1 infection.

Infection of CD127+ (interleukin-7 receptor+) CD4+ cells and overexpression of CTLA-4 are linked to loss of antigen-specific CD4 T cells during primary human immunodeficiency virus type 1 infection.

We recently found that human immunodeficiency virus (HIV)-specific CD4+ T cells express coreceptor CCR5 and activation antigen CD38 during early primary HIV-1 infection (PHI) but then rapidly disappear from the circulation. This cell loss may be due to susceptibility to infection with HIV-1 but could also be due to inappropriate apoptosis, an expansion of T regulatory cells, trafficking out of the circulation, or dysfunction. We purified CD38+++CD4+ T cells from peripheral blood mononuclear cells, measured their level of HIV-1 DNA by PCR, and found that about 10% of this population was infected. However, a small subset of HIV-specific CD4+) T cells also expressed CD127, a marker of long-term memory cells. Purified CD127+CD4+ lymphocytes contained fivefold more copies of HIV-1 DNA per cell than did CD127-negative CD4+ cells, suggesting preferential infection of long-term memory cells. We observed no apoptosis of antigen-specific CD4+ T cells in vitro and only a small increase in CD45RO+CD25+CD127dimCD4+ T regulatory cells during PHI. However, 40% of CCR5+CD38+++ CD4+ T cells expressed gut-homing integrins, suggesting trafficking through gut-associated lymphoid tissue (GALT). Furthermore, 80% of HIV-specific CD4+ T cells expressed high levels of the negative regulator CTLA-4 in response to antigen stimulation in vitro, which was probably contributing to their inability to produce interleukin-2 and proliferate. Taken together, the loss of HIV-specific CD4+ T cells is associated with a combination of an infection of CCR5+ CD127+ memory CD4+ T cells, possibly in GALT, and a high expression of the inhibitory receptor CTLA-4.

Sexual behaviour and human herpesvirus 8 infection in homosexual men in Australia.

BACKGROUND: Human herpesvirus 8 (HHV-8) is a common sexually transmitted agent among homosexual men, but there are few Australian data. We aimed to describe the prevalence and risk factors for seropositivity to HHV-8 in Australian homosexual men. METHODS: We conducted a prospective cohort study of 179 homosexual men in Sydney Australia in 1992-1998. Detailed data on sexual behaviour was collected annually, and HHV-8 status was determined at the end of the study by an algorithm based on results of an immunofluorescence assay and an enzyme-linked immunoassay to the K8.1 protein of HHV-8. HHV-8 DNA was detected in buffy coats using a nested qualitative PCR. RESULTS: Data on sexual behaviour in at least three interviews and HHV-8 status were available in 174 (97%) of 179 men who agreed to participate. Of these, 31 (18%) were HHV-8 seropositive, and HHV-8 DNA was detected in 5 (16%) of these. The prevalence of HHV-8 infection was much higher in HIV positive (52%) than HIV negative (11%) men (OR 8.60, 95% CI 3.55-20.86). HHV-8 infection was related to more frequent reporting of unprotected receptive anal sex (OR for most frequent versus least frequent category 3.03, 95% CI 1.01-9.03, P trend 0.02), insertive oro-anal sex (OR for most frequent v. least frequent category 3.02, 95% CI 1.15-7.93, P trend 0.02) and receptive oro-anal sex (OR for most frequent v. least frequent category 3.09, 95% CI 1.11-8.60, P trend 0.05) with casual partners. CONCLUSIONS: These data are consistent with sexual transmission of HHV-8, but the precise mode of HHV-8 transmission remains unclear. Studies to elucidate the precise mode of sexual transmission of HHV-8 need to focus on potential salivary transmission, and should collect data on the HHV-8 infection and excretion status of the sexual partner.

Early proliferation of CCR5(+) CD38(+++) antigen-specific CD4(+) Th1 effector cells during primary HIV-1 infection.

We investigated whether HIV-1 antigen-specific CD4(+) T cells expressed the viral coreceptor CCR5 during primary HIV-1 infection (PHI). In the peripheral blood of subjects with very early PHI (< 22 days after onset of symptoms), there was a 10- to 20-fold increase in the proportion of highly activated (CD38(+++)) and proliferating (Ki-67(+)) CD4(+) T cells that expressed CCR5(+), and were mostly T-cell intracellular antigen-1 (TIA-1)(+) perforin(+) granzyme B(+). Inthe same patient samples, CD4(+) T cells producing interferon (IFN)-gamma in response to HIV group-specific antigen (Gag) peptides were readily detected (median, 0.58%) by intracellular cytokine assay-these cells were again predominantly CD38(+++), Ki-67(+), and TIA-(++), as well as Bcl-2(low). On average, 20% of the Gag-specific CD4(+) T cells also expressed interleukin-2 (IL-2) and were CD127 (IL-7R)(+). Taken together, these results suggest that Gag-specific T-helper 1 (Th1) effector cells express CCR5 during the primary response and may include precursors of long-term self-renewing memory cells. However, in PHI subjects with later presentation, antigen-specific CD4(+) T cells could not be readily detected (median, 0.08%), coinciding with a 5-fold lower level of the CCR5(+)CD38(+++) CD4(+) T cells. These results suggest that the antiviral response to HIV-1 infection includes highly activated CCR5(+)CD4(+) cytotoxic effector cells, which are susceptible to both apoptosis and cytopathic infection with HIV-1, and rapidly decline.

Identification of circulating antigen-specific CD4+ T lymphocytes with a CCR5+, cytotoxic phenotype in an HIV-1 long-term nonprogressor and in CMV infection.

Antigen-specific CD4+ effector T cells primarily provide help for B-cell antibody responses and CD8+ cytotoxic T-lymphocyte (CTL) responses. We have found an expanded population of HIV-1 p24-specific, T-cell receptor V beta 17+, CD4+ T lymphocytes, defined by in vitro proliferative and interferon-gamma responses to a 15-mer Gag peptide, in the peripheral blood of an individual with long-term nonprogressive HIV-1 infection. Ex vivo, these cells were CCR5+ and CCR7-, consistent with an effector/memory function. Surprisingly, these cells highly expressed several proteins characteristic of cytotoxic lymphocytes, including TIA-1 (T-cell intracellular antigen 1; GMP-17/NKG7), granzymes A and B, CD161 (NKRP-1), and CD244 (C1.7/2B4). Following in vitro peptide stimulation, these cells produced interleukin 2 (IL-2) and intracellular CD40L, suggesting possible helper function, in addition to induction of perforin and cytotoxicity. A subset of cytomegalovirus (CMV)-specific CD4+ T cells in healthy adults similarly expressed these CTL markers and CCR5, ex vivo. Furthermore, this distinct subset of CD4+ T cells was significantly elevated in healthy CMV-seropositive adults, compared with CMV-seronegative individuals. These results suggest that CCR5+ CD4+ CTL may be a major effector mechanism of the immune response to viral infections in humans. Moreover, expression of CCR5 may render them particularly susceptible to cytopathic effects during progressive HIV-1 infection.