Virological, innate, and adaptive immune profiles shaped by variation in route and age of host in murine cytomegalovirus infection.

Human cytomegalovirus (hCMV) is a ubiquitous facultative pathogen, which establishes a characteristic latent and reactivating lifelong infection in immunocompetent hosts. Murine CMV (mCMV) infection is widely used as an experimental model of hCMV infection, employed to investigate the causal nature and extent of CMV's contribution to inflammatory, immunological, and health disturbances in humans. Therefore, mimicking natural human infection in mice would be advantageous to hCMV research. To assess the role of route and age at infection in modeling hCMV in mice, we infected prepubescent and young sexually mature C57BL/6 (B6) mice intranasally (i.n., a likely physiological route in humans) and intraperitoneally (i.p., a frequently used experimental route, possibly akin to transplant-mediated infection). In our hands, both routes led to comparable early viral loads and tissue spreads. However, they yielded differential profiles of innate and adaptive systemic immune activation. Specifically, the younger, prepubescent mice exhibited the strongest natural killer cell activation in the blood in response to i.p. infection. Further, the i.p. infected animals (particularly those infected at 12 weeks) exhibited larger anti-mCMV IgG and greater expansion of circulating CD8+ T cells specific for both acute (non-inflationary) and latent phase (inflationary) mCMV epitopes. By contrast, tissue immune responses were comparable between i.n. and i.p. groups. Our results illustrate a distinction in the bloodborne immune response profiles across infection routes and ages and are discussed in light of physiological parameters of interaction between CMV, immunity, inflammation, and health over the lifespan. IMPORTANCE: The majority of experiments modeling human cytomegalovirus (hCMV) infection in mice have been carried out using intraperitoneal infection in sexually mature adult mice, which stands in contrast to the large number of humans being infected with human CMV at a young age, most likely via bodily fluids through the nasopharyngeal/oral route. This study examined the impact of the choice of age and route of infection in modeling CMV infection in mice. By comparing young, prepubescent to older sexually mature counterparts, infected either via the intranasal or intraperitoneal route, we discovered substantial differences in deployment and response intensity of different arms of the immune system in systemic control of the virus; tissue responses, by contrast, appeared similar between ages and infection routes.

Anti-cytomegalovirus antibody levels stratify human immune profiles across the lifespan.

Human cytomegalovirus (hCMV) is a ubiquitous latent persistent herpesvirus infecting 60-90% of the population worldwide. hCMV carriage in immunocompetent people is asymptomatic; thus, hCMV can be considered a component of normative aging. However, hCMV powerfully modulates many features of the immune, and likely other, systems and organs. Questions remain as to how hCMV carriage affects the human host. We used anti-CMV antibody titers as a stratifying criterion to examine the impact of "intensity" of hCMV infection as a potential biomarker of aging, inflammation, and immune homeostasis in a cohort of 247 participants stratified into younger (21-40 years) and older (> 65 years of age) groups. We showed that anti-CMV antibody titers increased with age and directly correlated to increased levels of soluble tumor necrosis factor (sTNFR) I in younger but not older participants. CD8 + cell numbers were reduced in the older group due to the loss in CD8 + T naïve (Tn) cells. In CMV carriers and, in particular, in anti-CMV Ab-high participants, this loss was mitigated or reversed by an increase in the numbers of CD8 + T effector memory (Tem) and T effector memory reexpressing CD45RA (Temra) cells. Analysis of CD38, HLA-DR, and CD57 expression revealed subset (CD4 or CD8)-specific changes that correlated with anti-CMV Ab levels. In addition, anti-CMV Ab levels predicted anti-CMV CD8 T cell responsiveness to different CMV open reading frames (ORFs) selectively in older participants, which correlated to the transcriptional order of expression of specific CMV ORFs. Implications of these results for the potential predictive value of anti-CMV Ab titers during aging are discussed.

T-cell cellular stress and reticulocyte signatures, but not loss of naïve T lymphocytes, characterize severe COVID-19 in older adults.

In children and younger adults up to 39 years of age, SARS-CoV-2 usually elicits mild symptoms that resemble the common cold. Disease severity increases with age starting at 30 and reaches astounding mortality rates that are ~330 fold higher in persons above 85 years of age compared to those 18-39 years old. To understand age-specific immune pathobiology of COVID-19, we have analyzed soluble mediators, cellular phenotypes, and transcriptome from over 80 COVID-19 patients of varying ages and disease severity, carefully controlling for age as a variable. We found that reticulocyte numbers and peripheral blood transcriptional signatures robustly correlated with disease severity. By contrast, decreased numbers and proportion of naïve T-cells, reported previously as a COVID-19 severity risk factor, were found to be general features of aging and not of COVID-19 severity, as they readily occurred in older participants experiencing only mild or no disease at all. Single-cell transcriptional signatures across age and severity groups showed that severe but not moderate/mild COVID-19 causes cell stress response in different T-cell populations, and some of that stress was unique to old severe participants, suggesting that in severe disease of older adults, these defenders of the organism may be disabled from performing immune protection. These findings shed new light on interactions between age and disease severity in COVID-19.

Inflammatory and immune markers in HIV-infected older adults on long-term antiretroviral therapy: Persistent elevation of sCD14 and of proinflammatory effector memory T cells.

HIV-positive patients whose viral loads are successfully controlled by active antiretroviral therapy (ART) show no clinical signs of AIDS. However, their lifespan is shorter compared with individuals with no HIV infection and they prematurely exhibit a multitude of chronic diseases typically associated with advanced age. It was hypothesized that immune system aging may correlate with, and provide useful biomarkers for, this premature loss of healthspan in HIV-positive subjects. Here, we tested whether the immune correlates of aging, including cell numbers and phenotypes, inflammatory status, and control of human cytomegalovirus (hCMV) in HIV-positive subjects on long-term successful ART (HIV+) may reveal increased "immunological age" compared with HIV-negative, age-matched cohort (HIV-) in participants between 50 and 69 years of age. Specifically, we expected that younger HIV+ subjects may immunologically resemble older individuals without HIV. We found no evidence to support this hypothesis. While T cells from HIV+ participants displayed differential expression in several differentiation and/or inhibitory/exhaustion markers in different T cell subpopulations, aging by a decade did not pronounce these changes. Similarly, while the HIV+ participants exhibited higher T cell responses and elevated inflammatory marker levels in plasma, indicative of chronic inflammation, this trait was not age-sensitive. We did find differences in immune control of hCMV, and, more importantly, a sustained elevation of sCD14 and of proinflammatory CD4 and CD8 T cell responses across age groups, pointing towards uncontrolled inflammation as a factor in reduced healthspan in successfully treated older HIV+ patients.

Competent immune responses to SARS-CoV-2 variants in older adults following two doses of mRNA vaccination.

Aging is associated with a reduced magnitude of primary immune responses to vaccination. mRNA-based SARS-CoV-2 vaccines have shown efficacy in older adults but virus variant escape is still unclear. Here we analyze humoral and cellular immunity against an early-pandemic viral isolate and compare that to the P.1 (Gamma) and B.1.617.2 (Delta) variants in two cohorts (<50 and >55 age) of mRNA vaccine recipients. We further measure neutralizing antibody titers for B.1.617.1 (Kappa) and B.1.595, with the latter SARS-CoV-2 isolate bearing the spike mutation E484Q. Robust humoral immunity is measured following second vaccination, and older vaccinees manifest cellular immunity comparable to the adult group against early-pandemic SARS-CoV-2 and more recent variants. More specifically, the older cohort has lower neutralizing capacity at 7-14 days following the second dose but equilibrates with the younger cohort after 2-3 months. While long-term vaccination responses remain to be determined, our results implicate vaccine-induced protection in older adults against SARS-CoV-2 variants and inform thinking about boost vaccination.

Cutting Edge: T Cell Responses to B.1.1.529 (Omicron) SARS-CoV-2 Variant Induced by COVID-19 Infection and/or mRNA Vaccination Are Largely Preserved.

Several studies have demonstrated that the SARS-CoV-2 variant-of-concern B.1.1.529 (Omicron) exhibits a high degree of escape from Ab neutralization. Therefore, it is critical to determine how well the second line of adaptive immunity, T cell memory, performs against Omicron. To this purpose, we analyzed a human cohort (n = 327 subjects) of two- or three-dose mRNA vaccine recipients and COVID-19 postinfection subjects. We report that T cell responses against Omicron were largely preserved. IFN-γ-producing T cell responses remained equivalent to the response against the ancestral strain (WA1/2020), with some (∼20%) loss in IL-2 single or IL-2+IFN-γ+ polyfunctional responses. Three-dose vaccinated participants had similar responses to Omicron relative to post-COVID-19 participants and exhibited responses significantly higher than those receiving two mRNA vaccine doses. These results provide further evidence that a three-dose vaccine regimen benefits the induction of optimal functional T cell immune memory.

Early age-related atrophy of cutaneous lymph nodes precipitates an early functional decline in skin immunity in mice with aging.

Secondary lymphoid organs (SLOs) (including the spleen and lymph nodes [LNs]) are critical both for the maintenance of naive T (TN) lymphocytes and for the initiation and coordination of immune responses. How they age, including the exact timing, extent, physiological relevance, and the nature of age-related changes, remains incompletely understood. We used "time stamping" to indelibly mark newly generated naive T cells (also known as recent thymic emigrants) (RTEs) in mice, and followed their presence, phenotype, and retention in SLOs. We found that SLOs involute asynchronously. Skin-draining LNs atrophied by 6 to 9 mo in life, whereas deeper tissue-draining LNs atrophied by 18 to 20 mo, as measured by the loss of both TN numbers and the fibroblastic reticular cell (FRC) network. Time-stamped RTEs at all ages entered SLOs and successfully completed postthymic differentiation, but the capacity of older SLOs to maintain TN numbers was reduced with aging, and that trait did not depend on the age of TNs. However, in SLOs of older mice, these cells exhibited an emigration phenotype (CCR7loS1P1hi), which correlated with an increase of the cells of the same phenotype in the blood. Finally, upon intradermal immunization, RTEs generated in mice barely participated in de novo immune responses and failed to produce well-armed effector cells detectable in blood as early as by 7 to 8 mo of age. These results highlight changes in structure and function of superficial secondary lymphoid organs in laboratory mice that are earlier than expected and are consistent with the long-appreciated reduction of cutaneous immunity with aging.

Quantitative restoration of immune defense in old animals determined by naive antigen-specific CD8 T-cell numbers.

Older humans and animals often exhibit reduced immune responses to infection and vaccination, and this often directly correlates to the numbers and frequency of naive T (Tn) cells. We found such a correlation between reduced numbers of blood CD8+ Tn cells and severe clinical outcomes of West Nile virus (WNV) in both humans naturally exposed to, and mice experimentally infected with, WNV. To examine possible causality, we sought to increase the number of CD8 Tn cells by treating C57BL/6 mice with IL-7 complexes (IL-7C, anti-IL-7 mAb bound to IL-7), shown previously to efficiently increase peripheral T-cell numbers by homeostatic proliferation. T cells underwent robust expansion following IL-7C administration to old mice increasing the number of total T cells (>fourfold) and NS4b:H-2Db -restricted antigen-specific CD8 T cells (twofold). This improved the numbers of NS4b-specific CD8 T cells detected at the peak of the response against WNV, but not survival of WNV challenge. IL-7C-treated old animals also showed no improvement in WNV-specific effector immunity (neutralizing antibody and in vivo T-cell cytotoxicity). To test quantitative limits to which CD8 Tn cell restoration could improve protective immunity, we transferred graded doses of Ag-specific precursors into old mice and showed that injection of 5400 (but not of 1800 or 600) adult naive WNV-specific CD8 T cells significantly increased survival after WNV. These results set quantitative limits to the level of Tn reconstitution necessary to improve immune defense in older organisms and are discussed in light of targets of immune reconstitution.

Comparison of Chicken Immune Responses to Immunization with Vaccine La Sota or ZG1999HDS Strain of Newcastle Disease Virus.

Newcastle disease (ND) is a highly contagious avian disease. Global control of ND is mainly based on vaccination of poultry; however, reported outbreaks of ND in vaccinated flocks indicate a constant need to re-evaluate the existing vaccines and a development of the new ones. In this study, 4-week-old male chickens of the layer commercial hybrid were immunized oculonasally with a commercial NDV live La Sota vaccine (LS group), a suspension of lyophilized NDV strain ZG1999HDS (ZG group), or saline (Control (K) group). Antibody response was determined by haemagglutination inhibition (HI) assay. Cell-mediated immunity (CMI) was characterized by immunophenotyping of leukocyte's and T-lymphocyte's subpopulations (flow cytometry). Applied NDV strains did not cause any adverse reaction in treated chickens. Both strains induced the significantly higher HI antibody response in comparison to the control group, and overall antibody titer was higher in ZG group than in LS group. CMI, manifested as a higher proliferation of B- and T-helper cells, yielded better results in the ZG groups than in the LS group. Based on the obtained results, we conclude that the strain ZG1999HDS is immunogenic and is a suitable candidate for further research and development of poultry vaccines.

T-cell cellular stress and reticulocyte signatures, but not loss of naïve T lymphocytes, characterize severe COVID-19 in older adults.

In children and younger adults up to 39 years of age, SARS-CoV-2 usually elicits mild symptoms that resemble the common cold. Disease severity increases with age starting at 30 and reaches astounding mortality rates that are ~330 fold higher in persons above 85 years of age compared to those 18-39 years old. To understand age-specific immune pathobiology of COVID-19 we have analyzed soluble mediators, cellular phenotypes, and transcriptome from over 80 COVID-19 patients of varying ages and disease severity, carefully controlling for age as a variable. We found that reticulocyte numbers and peripheral blood transcriptional signatures robustly correlated with disease severity. By contrast, decreased numbers and proportion of naïve T-cells, reported previously as a COVID-19 severity risk factor, were found to be general features of aging and not of COVID-19 severity, as they readily occurred in older participants experiencing only mild or no disease at all. Single-cell transcriptional signatures across age and severity groups showed that severe but not moderate/mild COVID-19 causes cell stress response in different T-cell populations, and some of that stress was unique to old severe participants, suggesting that in severe disease of older adults, these defenders of the organism may be disabled from performing immune protection. These findings shed new light on interactions between age and disease severity in COVID-19.

Immune responses to two and three doses of the BNT162b2 mRNA vaccine in adults with solid tumors.

Vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have shown high efficacy, but immunocompromised participants were excluded from controlled clinical trials. In this study, we compared immune responses to the BNT162b2 mRNA Coronavirus Disease 2019 vaccine in patients with solid tumors (n = 53) who were on active cytotoxic anti-cancer therapy to a control cohort of participants without cancer (n = 50). Neutralizing antibodies were detected in 67% of patients with cancer after the first immunization, followed by a threefold increase in median titers after the second dose. Similar patterns were observed for spike protein-specific serum antibodies and T cells, but the magnitude of each of these responses was diminished relative to the control cohort. In most patients with cancer, we detected spike receptor-binding domain and other S1-specific memory B cell subsets as potential predictors of anamnestic responses to additional immunizations. We therefore initiated a phase 1 trial for 20 cancer cohort participants of a third vaccine dose of BNT162b2 ( NCT04936997 ); primary outcomes were immune responses, with a secondary outcome of safety. At 1 week after a third immunization, 16 participants demonstrated a median threefold increase in neutralizing antibody responses, but no improvement was observed in T cell responses. Adverse events were mild. These results suggest that a third dose of BNT162b2 is safe, improves humoral immunity against SARS-CoV-2 and could be immunologically beneficial for patients with cancer on active chemotherapy.

Infection-induced type I interferons critically modulate the homeostasis and function of CD8+ naïve T cells.

Naïve T (Tn) cells require two homeostatic signals for long-term survival: tonic T cell receptor:self-peptide-MHC contact and IL-7 stimulation. However, how microbial exposure impacts Tn homeostasis is still unclear. Here we show that infections can lead to the expansion of a subpopulation of long-lived, Ly6C+ CD8+ Tn cells with accelerated effector function. Mechanistically, mono-infection with West Nile virus transiently, and polymicrobial exposure persistently, enhances Ly6C expression selectively on CD5hiCD8+ cells, which in the case of polyinfection translates into a numerical CD8+ Tn cell increase in the lymph nodes. This conversion and expansion of Ly6C+ Tn cells depends on IFN-I, which upregulates MHC class I expression and enhances tonic TCR signaling in differentiating Tn cells. Moreover, for Ly6C+CD8+ Tn cells, IFN-I-mediated signals optimize their homing to secondary sites, extend their lifespan, and enhance their effector differentiation and antibacterial function, particularly for low-affinity clones. Our results thus uncover significant regulation of Tn homeostasis and function via infection-driven IFN-I, with potential implications for immunotherapy.

Immune response to COVID-19 in older adults.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the third highly pathogenic coronavirus to emerge in the human population in last two decades. SARS-CoV-2 spread from Wuhan, China, across the globe, causing an unprecedented public healthcare crisis. The virus showed remarkable age dependent pathology, with symptoms resembling common cold in most adults and children while causing more severe respiratory distress and significant mortality in older and frail humans. Even before the SARS-CoV-2 outbreak infectious diseases represented one of the major causes of death of older adults. Loss of immune function and reduced protection from infectious agents with age - immunosenescence - is a result of complex mechanisms affecting production and maintenance of immune cells as well as the initiation, maintenance and termination of properly directed immune responses. Here we briefly discuss the current knowledge on how this process affects age-dependent outcomes of SARS-CoV-2 infection.

Immune Responses to COVID-19 mRNA Vaccines in Patients with Solid Tumors on Active, Immunosuppressive Cancer Therapy.

Vaccines against SARS-CoV-2 have shown high efficacy, but immunocompromised participants were excluded from controlled clinical trials. We compared immune responses to the Pfizer/BioNTech mRNA vaccine in solid tumor patients (n=53) on active cytotoxic anti-cancer therapy to a control cohort (n=50) as an observational study. Using live SARS-CoV-2 assays, neutralizing antibodies were detected in 67% and 80% of cancer patients after the first and second immunizations, respectively, with a 3-fold increase in median titers after the booster. Similar trends were observed in serum antibodies against the receptor-binding domain (RBD) and S2 regions of Spike protein, and in IFNγ+ Spike-specific T cells. Yet the magnitude of each of these responses was diminished relative to the control cohort. We therefore quantified RBD- and Spike S1-specific memory B cell subsets as predictors of anamnestic responses to additional immunizations. After the second vaccination, Spike-specific plasma cell-biased memory B cells were observed in most cancer patients at levels similar to those of the control cohort after the first immunization. We initiated an interventional phase 1 trial of a third booster shot (NCT04936997); primary outcomes were immune responses with a secondary outcome of safety. After a third immunization, the 20 participants demonstrated an increase in antibody responses, with a median 3-fold increase in virus-neutralizing titers. Yet no improvement was observed in T cell responses at 1 week after the booster immunization. There were mild adverse events, primarily injection site myalgia, with no serious adverse events after a month of follow-up. These results suggest that a third vaccination improves humoral immunity against COVID-19 in cancer patients on active chemotherapy with no severe adverse events.

Immunogenicity of Newcastle disease virus strain ZG1999HDS applied oculonasally or by means of nebulization to day-old chicks.

Newcastle disease (ND) is one of the classic viral infections of poultry which resists all the efforts of eradication. Newcastle disease virus (NDV) strain ZG1999HDS was isolated during the outbreak in 1,999 at a broiler farm in Croatia. Previous trials in chickens confirmed it to be a lentogenic pathotype and immunogenic by stimulating humoral and cell mediated immunity. Further characterization by deduced amino acid sequence at the cleavage site of fusion protein confirmed its lentogenic nature, and in vitro tests its oncolytic capacity. Owing to its immunogenicity, strain ZG1999HDS is considered for vaccine development. In this study, 1-day-old chicks were vaccinated using strain ZG1999HDS oculonasally or by nebulization. Strain ZG1999HDS induced humoral immune response in both immunized groups The cell-mediated immune response occurred earlier in the group immunized by nebulization, as shown by a higher frequency rate of T and B lymphocytes, and significantly higher expression of IFN-α in respiratory organs and IFN-γ expression in the spleen. Viral genomic RNA was not detected in investigated organs. Thus, NDV strain ZG1999HDS is immunogenic when administered by means of nebulization or oculonasally without any adverse effects and is therefore suitable for further research and vaccine development. Further research is needed regarding its tropism.

IL-6 can singlehandedly drive many features of frailty in mice.

Frailty is a geriatric syndrome characterized by age-related declines in function and reserve resulting in increased vulnerability to stressors. The most consistent laboratory finding in frail subjects is elevation of serum IL-6, but it is unclear whether IL-6 is a causal driver of frailty. Here, we characterize a new mouse model of inducible IL-6 expression (IL-6TET-ON/+ mice) following administration of doxycycline (Dox) in food. In this model, IL-6 induction was Dox dose-dependent. The Dox dose that increased IL-6 levels to those observed in frail old mice directly led to an increase in frailty index, decrease in grip strength, and disrupted muscle mitochondrial homeostasis. Littermate mice lacking the knock-in construct failed to exhibit frailty after Dox feeding. Both naturally old mice and young Dox-induced IL-6TET-ON/+ mice exhibited increased IL-6 levels in sera and spleen homogenates but not in other tissues. Moreover, Dox-induced IL-6TET-ON/+ mice exhibited selective elevation in IL-6 but not in other cytokines. Finally, bone marrow chimera and splenectomy experiments demonstrated that non-hematopoietic cells are the key source of IL-6 in our model. We conclude that elevated IL-6 serum levels directly drive age-related frailty, possibly via mitochondrial mechanisms.

Corrigendum: Defective Transcriptional Programming of Effector CD8 T Cells in Aged Mice Is Cell-Extrinsic and Can Be Corrected by Administration of IL-12 and IL-18.

[This corrects the article DOI: 10.3389/fimmu.2019.02206.].

Defective Transcriptional Programming of Effector CD8 T Cells in Aged Mice Is Cell-Extrinsic and Can Be Corrected by Administration of IL-12 and IL-18.

In response to infection with intracellular microorganisms, old mice mobilize decreased numbers of antigen-specific CD8 T cells with reduced expression of effector molecules and impaired cytolytic activity. Molecular mechanisms behind these defects and the cell-intrinsic (affecting naïve CD8 T cells themselves) vs. extrinsic, microenvironmental origin of such defects remain unclear. Using reciprocal transfer experiments of highly purified naïve T cells from adult and old transgenic OT-1 mice, we decisively show that the dominant effect is cell-extrinsic. Naïve adult OT-1 T cells failed to expand and terminally differentiate in the old organism infected with Listeria-OVA. This defect was preceded by blunted expression of the master transcription factor T-bet and impaired glycolytic switch when T cells are primed in the old organism. However, both old and adult naïve CD8 T cells proliferated and produced effector molecules to a similar extent when stimulated in vitro with polyclonal stimuli, as well as when transferred into adult recipients. Multiple inflammatory cytokines with direct effects on T cell effector differentiation were decreased in spleens of old animals, particularly IL-12 and IL-18. Of note, in vivo treatment of mice with IL-12 and IL-18 on days 4-6 of Listeria infection reconstituted cytotoxic T cell response of aged mice to the level of adult. Therefore, critical cytokine signals which are underproduced in the old priming environment can restore proper transcriptional programming of old naïve CD8 T cells and improve immune defense against intracellular microorganisms.

Do cytomegalovirus-specific memory T cells interfere with new immune responses in lymphoid tissues?

In both mice and humans, the CD8 T cell compartment is expanded with age in the presence of a cytomegalovirus (CMV) infection due to an absolute increase in the CD8+ T cell effector memory (TEM) cells. It has been hypothesized that in CMV+ subjects, such accumulated TEM cells could interfere with responses to new infection by competing for space/resources or could inhibit new responses by other, undefined, means. Here we present evidence against this hypothesis. We show that MCMV-specific CD8 T cells accumulate in blood and bone marrow, but not lymph nodes (frequent sites of immune response initiation), in either persistent lifelong CMV infection or following reactivation. Moreover, adoptive transfer of effector memory T cells from MCMV positive mice into naïve animals did not interfere with either humoral or cellular response to West Nile virus or Listeria monocytogenes infection in recipient mice. We conclude that MCMV infection is unlikely to inhibit new immune responses in old animals through direct interference of MCMV-specific CD8 T cells with the priming.