Diminished humoral and cellular responses to SARS-CoV-2 vaccines in patients with chronic lymphocytic leukemia.

Previous studies have demonstrated low rates of seroconversion to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) messenger RNA (mRNA) vaccines in patients with chronic lymphocytic leukemia (CLL). In this national collaboration of 11 cancer centers in the United States, we aimed to further characterize and understand vaccine-induced immune responses, including T-cell responses, and the impact of CLL therapeutics (#NCT04852822). Eligible patients were enrolled in 2 cohorts (1) at the time of initial vaccination and (2) at the time of booster vaccination. The serologic response rates (anti-S) from 210 patients in the initial vaccination cohort and 117 in the booster vaccination cohort were 56% (95% confidence interval [CI], 50-63) and 68% (95% CI, 60-77), respectively. Compared with patients not on therapy, those receiving B-cell-directed therapy were less likely to seroconvert (odds ratio [OR], 0.27; 95% CI, 0.15-0.49). Persistence of response was observed at 6 months; anti-S titers increased with the administration of booster vaccinations. In the initial vaccination cohort, positive correlations were observed between the quantitative serologic response and CD4 T-cell response for the Wuhan variant and, to a lesser degree, for the Omicron variant (Spearman P = 0.45 Wuhan; P = 0.25 Omicron). In the booster vaccination cohort, positive correlations were observed between serologic responses and CD4 T-cell responses for both variants (P = 0.58 Wuhan; P = 0.57 Omicron) and to a lesser degree for CD8 T-cell responses (P = 0.33 Wuhan; P = 0.22 Omicron). Although no deaths from coronavirus disease 2019 (COVID-19) have been reported after booster vaccinations, patients should use caution as newer variants emerge and escape vaccine-induced immunity. This trial was registered at www.clinicaltrials.gov as #NCT04852822.

HLA-B*57:01 Complexed to a CD8 T-Cell Epitope from the HSV-2 ICP22 Protein Binds NK and T Cells through KIR3DL1.

HLA-B*57:01 is an HLA allelic variant associated with positive outcomes during viral infections through interactions with T cells and NK cells, but severe disease in persons treated with the anti-HIV-1 drug abacavir. The role of HLA-B*57:01 in the context of HSV infection is unknown. We identified an HLA-B*57:01-restricted CD8 T-cell epitope in the ICP22 (US1) protein of HSV-2. CD8 T cells reactive to the HSV-2 ICP22 epitope recognized the orthologous HSV-1 peptide, but not closely related peptides in human IFNL2 or IFNL3. Abacavir did not alter the CD8 T-cell recognition of the HSV or self-derived peptides. Unexpectedly, a tetramer of HSV-2 ICP22 epitope (228-236) and HLA-B*57:01 bound both CD8 T cells and NK cells. Tetramer specificity for KIR3DL1 was confirmed using KIR3DL1 overexpression on non-human primate cells lacking human KIR and studies with blocking anti-KIR3DL1 antibody. Interaction with KIR3DL1 was generalizable to donors lacking the HLA-B*57:01 genotype or HSV seropositivity. These findings suggest a mechanism for the recognition of HSV infection by NK cells or KIR-expressing T cells via KIR3DL1.

HSV-2-Specific Human Female Reproductive Tract Tissue Resident Memory T Cells Recognize Diverse HSV Antigens.

Antigen-specific TRM persist and protect against skin or female reproductive tract (FRT) HSV infection. As the pathogenesis of HSV differs between humans and model organisms, we focus on humans with well-characterized recurrent genital HSV-2 infection. Human CD8+ TRM persisting at sites of healed human HSV-2 lesions have an activated phenotype but it is unclear if TRM can be cultivated in vitro. We recovered HSV-specific TRM from genital skin and ectocervix biopsies, obtained after recovery from recurrent genital HSV-2, using ex vivo activation by viral antigen. Up to several percent of local T cells were HSV-reactive ex vivo. CD4 and CD8 T cell lines were up to 50% HSV-2-specific after sorting-based enrichment. CD8 TRM displayed HLA-restricted reactivity to specific HSV-2 peptides with high functional avidities. Reactivity to defined peptides persisted locally over several month and was quite subject-specific. CD4 TRM derived from biopsies, and from an extended set of cervical cytobrush specimens, also recognized diverse HSV-2 antigens and peptides. Overall we found that HSV-2-specific TRM are abundant in the FRT between episodes of recurrent genital herpes and maintain competency for expansion. Mucosal sites are accessible for clinical monitoring during immune interventions such as therapeutic vaccination.

BTK inhibitors impair humoral and cellular responses to recombinant zoster vaccine in CLL.

Vaccinations effectively prevent infections; however, patients with chronic lymphocytic leukemia (CLL) have reduced antibody responses following vaccinations. Combined humoral and cellular immune responses to novel adjuvanted vaccines are not well characterized in CLL. In an open-label, single-arm clinical trial, we measured the humoral and cellular immunogenicity of the recombinant zoster vaccine (RZV) in CLL patients who were treatment naïve (TN) or receiving Bruton tyrosine kinase inhibitor (BTKi) therapy. The primary endpoint was antibody response to RZV (≥fourfold increase in anti-glycoprotein E [anti-gE]). Cellular response of gE-specific CD4+ T cells was assessed by flow cytometry for upregulation of ≥2 effector molecules. The antibody response rate was significantly higher in the TN cohort (76.8%; 95% confidence interval [CI], 65.7-87.8) compared with patients receiving a BTKi (40.0%; 95% CI, 26.4-53.6; P = .0002). The cellular response rate was also significantly higher in the TN cohort (70.0%; 95% CI, 57.3-82.7) compared with the BTKi group (41.3%; 95% CI, 27.1-55.5; P = .0072). A concordant positive humoral and cellular immune response was observed in 69.1% (95% CI, 56.9-81.3) of subjects with a humoral response, whereas 39.0% (95% CI, 24.1-54.0) of subjects without a humoral response attained a cellular immune response (P = .0033). Antibody titers and T-cell responses were not correlated with age, absolute B- and T-cell counts, or serum immunoglobulin levels (all P > .05). RZV induced both humoral and cellular immune responses in treated and untreated CLL patients, albeit with lower response rates in patients on BTKi therapy compared with TN patients. This trial was registered at www.clinicaltrials.gov as #NCT03702231.

Human CD4+ T Cells Specific for Merkel Cell Polyomavirus Localize to Merkel Cell Carcinomas and Target a Required Oncogenic Domain.

Although CD4+ T cells likely play key roles in antitumor immune responses, most immuno-oncology studies have been limited to CD8+ T-cell responses due to multiple technical barriers and a lack of shared antigens across patients. Merkel cell carcinoma (MCC) is an aggressive skin cancer caused by Merkel cell polyomavirus (MCPyV) oncoproteins in 80% of cases. Because MCPyV oncoproteins are shared across most patients with MCC, it is unusually feasible to identify, characterize, and potentially augment tumor-specific CD4+ T cells. Here, we report the identification of CD4+ T-cell responses against six MCPyV epitopes, one of which included a conserved, essential viral oncogenic domain that binds/disables the cellular retinoblastoma (Rb) tumor suppressor. We found that this epitope (WEDLT209-228) could be presented by three population-prevalent HLA class II alleles, making it a relevant target in 64% of virus-positive MCC patients. Cellular staining with a WEDLT209-228-HLA-DRB1*0401 tetramer indicated that specific CD4+ T cells were detectable in 78% (14 of 18) of evaluable MCC patients, were 250-fold enriched within MCC tumors relative to peripheral blood, and had diverse T-cell receptor sequences. We also identified a modification of this domain that still allowed recognition by these CD4+ T cells but disabled binding to the Rb tumor suppressor, a key step in the detoxification of a possible therapeutic vaccine. The use of these new tools for deeper study of MCPyV-specific CD4+ T cells may provide broader insight into cancer-specific CD4+ T-cell responses.

Selective Expression of CCR10 and CXCR3 by Circulating Human Herpes Simplex Virus-Specific CD8 T Cells.

Herpes simplex virus (HSV) infection is restricted to epithelial cells and neurons and is controlled by CD8 T cells. These cells both traffic to epithelial sites of recurrent lytic infection and to ganglia and persist at the dermal-epidermal junction for up to 12 weeks after lesion resolution. We previously showed that cutaneous lymphocyte-associated antigen (CLA), a functional E-selectin ligand (ESL), is selectively expressed on circulating HSV-2-specific CD8 T cells. CLA/ESL mediates adhesion of T cells to inflamed vascular endothelium. Later stages in T-cell homing involve chemokines (Ch) and lymphocyte chemokine receptors (ChR) for vascular wall arrest and diapedesis. Several candidate ChR have been implicated in skin homing. We measured cell surface ChR on HSV-specific human peripheral blood CD8 T cells and extended our studies to HSV-1. We observed preferential cell surface expression of CCR10 and CXCR3 by HSV-specific CD8 T cells compared to CD8 T cells specific for control viruses, Epstein-Barr virus (EBV) and cytomegalovirus (CMV), and compared to bulk memory CD8 T cells. CXCR3 ligand mRNA levels were selectively increased in skin biopsy specimens from persons with recurrent HSV-2, while the mRNA levels of the CCR10 ligand CCL27 were equivalent in lesion and control skin. Our data are consistent with a model in which CCL27 drives baseline recruitment of HSV-specific CD8 T cells expressing CCR10, while interferon-responsive CXCR3 ligands recruit additional cells in response to virus-driven inflammation.IMPORTANCE HSV-2 causes very localized recurrent infections in the skin and genital mucosa. Virus-specific CD8 T cells home to the site of recurrent infection and participate in viral clearance. The exit of T cells from the blood involves the use of chemokine receptors on the T-cell surface and chemokines that are present in infected tissue. In this study, circulating HSV-2-specific CD8 T cells were identified using specific fluorescent tetramer reagents, and their expression of several candidate skin-homing-associated chemokine receptors was measured using flow cytometry. We found that two chemokine receptors, CXCR3 and CCR10, are upregulated on HSV-specific CD8 T cells in blood. The chemokines corresponding to these receptors are also expressed in infected tissues. Vaccine strategies to prime CD8 T cells to home to HSV lesions should elicit these chemokine receptors if possible to increase the homing of vaccine-primed cells to sites of infection.

Extensive CD4 and CD8 T Cell Cross-Reactivity between Alphaherpesviruses.

The Alphaherpesvirinae subfamily includes HSV types 1 and 2 and the sequence-divergent pathogen varicella zoster virus (VZV). T cells, controlled by TCR and HLA molecules that tolerate limited epitope amino acid variation, might cross-react between these microbes. We show that memory PBMC expansion with either HSV or VZV enriches for CD4 T cell lines that recognize the other agent at the whole-virus, protein, and peptide levels, consistent with bidirectional cross-reactivity. HSV-specific CD4 T cells recovered from HSV-seronegative persons can be explained, in part, by such VZV cross-reactivity. HSV-1-reactive CD8 T cells also cross-react with VZV-infected cells, full-length VZV proteins, and VZV peptides, as well as kill VZV-infected dermal fibroblasts. Mono- and cross-reactive CD8 T cells use distinct TCRB CDR3 sequences. Cross-reactivity to VZV is reconstituted by cloning and expressing TCRA/TCRB receptors from T cells that are initially isolated using HSV reagents. Overall, we define 13 novel CD4 and CD8 HSV-VZV cross-reactive epitopes and strongly imply additional cross-reactive peptide sets. Viral proteins can harbor both CD4 and CD8 HSV/VZV cross-reactive epitopes. Quantitative estimates of HSV/VZV cross-reactivity for both CD4 and CD8 T cells vary from 10 to 50%. Based on these findings, we hypothesize that host herpesvirus immune history may influence the pathogenesis and clinical outcome of subsequent infections or vaccinations for related pathogens and that cross-reactive epitopes and TCRs may be useful for multi-alphaherpesvirus vaccine design and adoptive cellular therapy.

Virologic and immunologic evidence of multifocal genital herpes simplex virus 2 infection.

UNLABELLED: Genital herpes simplex virus (HSV) reactivation is thought to be anatomically and temporally localized, coincident with limited ganglionic infection. Short, subclinical shedding episodes are the most common form of HSV-2 reactivation, with host clearance mechanisms leading to rapid containment. The anatomic distribution of shedding episodes has not been characterized. To precisely define patterns of anatomic reactivation, we divided the genital tract into a 22-region grid and obtained daily swabs for 20 days from each region in 28 immunocompetent, HSV-2-seropositive persons. HSV was detected via PCR, and sites of asymptomatic HSV shedding were subjected to a biopsy procedure within 24 h. CD4(+) and CD8(+) T cells were quantified by immunofluorescence, and HSV-specific CD4(+) T cells were identified by intracellular cytokine cytometry. HSV was detected in 868 (7%) of 11,603 genital swabs at a median of 12 sites per person (range, 0 to 22). Bilateral HSV detection occurred on 83 (67%) days with shedding, and the median quantity of virus detected/day was associated with the number of sites positive (P < 0.001). In biopsy specimens of asymptomatic shedding sites, we found increased numbers of CD8(+) T cells compared to control tissue (27 versus 13 cells/mm(2), P = 0.03) and identified HSV-specific CD4(+) T cells. HSV reactivations emanate from widely separated anatomic regions of the genital tract and are associated with a localized cellular infiltrate that was demonstrated to be HSV specific in 3 cases. These data provide evidence that asymptomatic HSV-2 shedding contributes to chronic inflammation throughout the genital tract. IMPORTANCE: This detailed report of the anatomic patterns of genital HSV-2 shedding demonstrates that HSV-2 reactivation can be detected at multiple bilateral sites in the genital tract, suggesting that HSV establishes latency throughout the sacral ganglia. In addition, genital biopsy specimens from sites of asymptomatic HSV shedding have increased numbers of CD8(+) T cells compared to control tissue, and HSV-specific CD4(+) T cells are found at sites of asymptomatic shedding. These findings suggest that widespread asymptomatic genital HSV-2 shedding is associated with a targeted host immune response and contributes to chronic inflammation throughout the genital tract.

Cross-presentation and genome-wide screening reveal candidate T cells antigens for a herpes simplex virus type 1 vaccine.

Herpes simplex virus type 1 (HSV-1) not only causes painful recurrent oral-labial infections, it can also cause permanent brain damage and blindness. There is currently no HSV-1 vaccine. An effective vaccine must stimulate coordinated T cell responses, but the large size of the genome and the low frequency of HSV-1-specific T cells have hampered the search for the most effective T cell antigens for inclusion in a candidate vaccine. We have now developed what we believe to be novel methods to efficiently generate a genome-wide map of the responsiveness of HSV-1-specific T cells, and demonstrate the applicability of these methods to a second complex microbe, vaccinia virus. We used cross-presentation and CD137 activation-based FACS to enrich for polyclonal CD8+ T effector T cells. The HSV-1 proteome was prepared in a flexible format for analyzing both CD8+ and CD4+ T cells from study participants. Scans with participant-specific panels of artificial APCs identified an oligospecific response in each individual. Parallel CD137-based CD4+ T cell research showed discrete oligospecific recognition of HSV-1 antigens. Unexpectedly, the two HSV-1 proteins not previously considered as vaccine candidates elicited both CD8+ and CD4+ T cell responses in most HSV-1-infected individuals. In this era of microbial genomics, our methods - also demonstrated in principle for vaccinia virus for both CD8+ and CD4+ T cells - should be broadly applicable to the selection of T cell antigens for inclusion in candidate vaccines for many pathogens.

Description of an elasmobranch TCR coreceptor: CD8α from Rhinobatos productus.

Cell-mediated immunity plays an essential role for the control and eradication of intracellular pathogens. To learn more about the evolutionary origins of the first signal (Signal 1) for T-cell activation, we cloned CD8α from an elasmobranch, Rhinobatos productus. Similar to full-length CD8α cDNAs from other vertebrates, Rhpr-CD8α (1800bp) encodes a 219 amino acid open reading frame composed of a signal peptide, an extracellular IgSF V domain and a stalk/hinge region followed by a well-conserved transmembrane domain and cytoplasmic tail. Overall, the mature Rhpr-CD8α protein (201 aa) displays ∼ 30% amino acid identity with mammalian CD8α including absolute conservation of cysteine residues involved in the IgSf V domain fold and dimerization of CD8αα and CD8αß. One prominent feature is the absence of the LCK association motif (CXC) that is needed for achieving signal 1 in tetrapods. Both elasmobranch and teleost CD8α protein sequences possess a similar but distinctly different motif (CXH) in the cytoplasmic tail. The overall genomic structure of CD8α has been conserved during the course of vertebrate evolution both for the number of exons and phase of splicing. Finally, quantitative RTPCR demonstrated that elasmobranch CD8α is expressed in lymphoid-rich tissues similar to CD8 in other vertebrates. The results from this study indicate the existence of CD8 prior to the emergence of the gnathostomes (>450 MYA) while providing evidence that the canonical LCK association motif in mammals is likely a derived characteristic of tetrapod CD8α, suggesting potential differences for T-cell education and activation in the various gnathostomes.

Characterization of the interferon genes in homozygous rainbow trout reveals two novel genes, alternate splicing and differential regulation of duplicated genes.

The genes encoding the type I and type II interferons (IFNs) have previously been identified in rainbow trout and their proteins partially characterized. These previous studies reported a single type II IFN (rtIFN-gamma) and three rainbow trout type I IFN genes that are classified into either group I (rtIFN1, rtIFN2) or group II (rtIFN3). In this present study, we report the identification of a novel IFN-gamma gene (rtIFN-gamma2) and a novel type I group II IFN (rtIFN4) in homozygous rainbow trout and predict that additional IFN genes or pseudogenes exist in the rainbow trout genome. Additionally, we provide evidence that short and long forms of rtIFN1 are actively and differentially transcribed in homozygous trout, and likely arose due to alternate splicing of the first exon. Quantitative reverse transcriptase PCR (qRT-PCR) assays were developed to systematically profile all of the rainbow trout IFN transcripts, with high specificity at an individual gene level, in naïve fish and after stimulation with virus or viral-related molecules. Cloned PCR products were used to ensure the specificity of the qRT-PCR assays and as absolute standards to assess transcript abundance of each gene. All IFN genes were modulated in response to Infectious hematopoietic necrosis virus (IHNV), a DNA vaccine based on the IHNV glycoprotein, and poly I:C. The most inducible of the type I IFN genes, by all stimuli tested, were rtIFN3 and the short transcript form of rtIFN1. Gene expression of rtIFN-gamma1 and rtIFN-gamma2 was highly up-regulated by IHNV infection and DNA vaccination but rtIFN-gamma2 was induced to a greater magnitude. The specificity of the qRT-PCR assays reported here will be useful for future studies aimed at identifying which cells produce IFNs at early time points after infection.

Molecular and biochemical analysis of rainbow trout LCK suggests a conserved mechanism for T-cell signaling in gnathostomes.

Two genes were identified in rainbow trout that display high sequence identity to vertebrate Lck. Both of the trout Lck transcripts are associated with lymphoid tissues and were found to be highly expressed in IgM-negative lymphocytes. In vitro analysis of trout lymphocytes indicates that trout Lck mRNA is up-regulated by T-cell mitogens, supporting an evolutionarily conserved function for Lck in the signaling pathways of T-lymphocytes. Here, we describe the generation and characterization of a specific monoclonal antibody raised against the N-terminal domains of recombinant trout Lck that can recognize Lck protein(s) from trout thymocyte lysates that are similar in size ( approximately 57kDa) to mammalian Lck. This antibody also reacted with permeabilized lymphocytes during FACS analysis, indicating its potential usage for cellular analyses of trout lymphocytes, thus representing an important tool for investigations of salmonid T-cell function.

Chemokines.

Chemokines are small proteins that control cellular migration. An extensive family of these molecules has been described in mammals containing nearly 50 members. Within this family are four groups, each defined by the different spacing of two N-terminal cysteines, which form disulphide bonds with two other cysteine residues to create the tertiary structure characteristic of chemokines. Recent evidence shows the chemokine family is not unique to mammals, with several members also identified in birds, amphibians and fish, including a primitive vertebrate, the lamprey. Although there is less evidence to define the roles of chemokines in these lower vertebrates, structural similarities allow some predictions to their function, against which further studies are being made. Additionally, some microorganisms (particularly viruses) appear to have copied genes for chemokines, presumably to confuse the immune system of their host. This review aims to bring together the current information concerning identified chemokines throughout vertebrates and microorganisms.

Identification and analysis of an interleukin 8-like molecule in rainbow trout Oncorhynchus mykiss.

An interleukin 8 (IL-8) homologue has been identified in the rainbow trout Oncorhynchus mykiss. The transcript contains an open reading frame of 294 nucleotides that translates into a 97 amino acid putative peptide, with 5' and 3' untranslated regions (UTR) of 171 and 453 nucleotides, respectively. As with previously sequenced lamprey and flounder genes, the trout amino acid sequence lacks the typical ELR motif upstream of the first pair of cysteines, where DLR is present. The trout IL-8 gene contains four exons divided by three short introns of 341, 247 and 292bp, and occupies 1824bp of genomic DNA. RT-PCR reveals a low level constitutive expression of the IL-8 homologue in many tissues, including spleen, heart, liver, head kidney and gill. Expression was not detectable in the brain. Whilst no apparent affect of lipopolysaccharide (LPS) on IL-8 expression was observed in vivo, stimulation of a trout macrophage cell line (RTS-11) with either LPS or poly I:C did result in clear up-regulation of IL-8 expression, detectable by RT-PCR and Northern blot analysis.