Distinct in vitro and in vivo neutralization profiles of monoclonal antibodies elicited by the receptor binding domain of the ancestral SARS-CoV-2.

Broadly neutralizing antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants are sought to curb coronavirus disease 2019 (COVID-19) infections. Here we produced and characterized a set of mouse monoclonal antibodies (mAbs) specific for the ancestral SARS-CoV-2 receptor binding domain (RBD). Two of them, 17A7 and 17B10, were highly potent in microneutralization assay with 50% inhibitory concentration (IC50 ) ≤135 ng/mL against infectious SARS-CoV-2 variants, including G614, Alpha, Beta, Gamma, Delta, Epsilon, Zeta, Kappa, Lambda, B.1.1.298, B.1.222, B.1.5, and R.1. Both mAbs (especially 17A7) also exhibited strong in vivo efficacy in protecting K18-hACE2 transgenic mice from the lethal infection with G614, Alpha, Beta, Gamma, and Delta viruses. Structural analysis indicated that 17A7 and 17B10 target the tip of the receptor binding motif in the RBD-up conformation. A third RBD-reactive mAb (3A6) although escaped by Beta and Gamma, was highly effective in cross-neutralizing Delta and Omicron BA.1 variants in vitro and in vivo. In competition experiments, antibodies targeting epitopes similar to these 3 mAbs were rarely enriched in human COVID-19 convalescent sera or postvaccination sera. These results are helpful to inform new antibody/vaccine design and these mAbs can be useful tools for characterizing SARS-CoV-2 variants and elicited antibody responses.

Enhanced virulence and waning vaccine-elicited antibodies account for breakthrough infections caused by SARS-CoV-2 delta and beyond.

Here we interrogate the factors responsible for SARS-CoV-2 breakthrough infections in a K18-hACE2 transgenic mouse model. We show that Delta and the closely related Kappa variant cause viral pneumonia and severe lung lesions in K18-hACE2 mice. Human COVID-19 mRNA post-vaccination sera after the 2nd dose are significantly less efficient in neutralizing Delta/Kappa than early 614G virus in vitro and in vivo. By 5 months post-vaccination, ≥50% of donors lack detectable neutralizing antibodies against Delta and Kappa and all mice receiving 5-month post-vaccination sera die after the lethal challenges. Although a 3rd vaccine dose can boost antibody neutralization against Delta in vitro and in vivo, the mean log neutralization titers against the latest Omicron subvariants are 1/3-1/2 of those against the original 614D virus. Our results suggest that enhanced virulence, greater immune evasion, and waning of vaccine-elicited protection account for SARS-CoV-2 variants caused breakthrough infections.

Post-Prandial Amino Acid Changes in Gilthead Sea Bream.

Following a meal, a series of physiological changes occurs in fish as they digest, absorb and assimilate ingested nutrients. This study aims to assess post-prandial free amino acid (FAA) activity in gilthead sea bream consuming a partial marine protein (fishmeal) replacement. Sea bream were fed diets where 16 and 27% of the fishmeal protein was replaced by plant protein. The essential amino acid (EAA) composition of the white muscle, liver and gut of sea bream was strongly correlated with the EAA composition of the 16% protein replacement diet compared to the 27% protein replacement diet. The mean FAA concentration in the white muscle and liver changed at 4 to 8 h after a meal and was not different to pre-feeding (0 h) and at 24 h after feeding. It was confirmed in this study that 16% replacement of marine protein with plant protein meets the amino acid needs of sea bream. Overall, the present study contributes towards understanding post-prandial amino acid profiles during uptake, tissue assimilation and immediate metabolic processing of amino acids in sea bream consuming a partial marine protein replacement. This study suggests the need to further investigate the magnitude of the post-prandial tissue-specific amino acid activity in relation to species-specific abilities to regulate metabolism due to dietary nutrient utilization.

Age-dependency of terminal ileum tissue resident memory T cell responsiveness profiles to S. Typhi following oral Ty21a immunization in humans.

BACKGROUND: The impact of aging on the immune system is unequivocal and results in an altered immune status termed immunosenescence. In humans, the mechanisms of immunosenescence have been examined almost exclusively in blood. However, most immune cells are present in tissue compartments and exhibit differential cell (e.g., memory T cells -TM) subset distributions. Thus, it is crucial to understand immunosenescence in tissues, especially those that are exposed to pathogens (e.g., intestine). Using a human model of oral live attenuated typhoid vaccine, Ty21a, we investigated the effect of aging on terminal ileum (TI) tissue resident memory T (TRM) cells. TRM provide immediate adaptive effector immune responsiveness at the infection site. However, it is unknown whether aging impacts TRM S. Typhi-responsive cells at the site of infection (e.g., TI). Here, we determined the effect of aging on the induction of TI S. Typhi-responsive TRM subsets elicited by Ty21a immunization. RESULTS: We observed that aging impacts the frequencies of TI-lamina propria mononuclear cells (LPMC) TM and TRM in both Ty21a-vaccinated and control groups. In unvaccinated volunteers, the frequencies of LPMC CD103- CD4+ TRM displayed a positive correlation with age whilst the CD4/CD8 ratio in LPMC displayed a negative correlation with age. We observed that elderly volunteers have weaker S. Typhi-specific mucosal immune responses following Ty21a immunization compared to adults. For example, CD103+ CD4+ TRM showed reduced IL-17A production, while CD103- CD4+ TRM exhibited lower levels of IL-17A and IL-2 in the elderly than in adults following Ty21a immunization. Similar results were observed in LPMC CD8+ TRM and CD103- CD8+ T cell subsets. A comparison of multifunctional (MF) profiles of both CD4+ and CD8+ TRM subsets between elderly and adults also showed significant differences in the quality and quantity of elicited single (S) and MF responses. CONCLUSIONS: Aging influences tissue resident TM S. Typhi-specific responses in the terminal ileum following oral Ty21a-immunization. This study is the first to provide insights in the generation of local vaccine-specific responses in the elderly population and highlights the importance of evaluating tissue immune responses in the context of infection and aging. TRIAL REGISTRATION: This study was approved by the Institutional Review Board and registered on ClinicalTrials.gov (identifier NCT03970304 , Registered 29 May 2019 - Retrospectively registered).

The SENIEUR protocol and the efficacy of hepatitis B vaccination in healthy elderly persons by age, gender, and vaccine route.

BACKGROUND: Reduced response to hepatitis B vaccines is associated with aging, confounding and comorbid conditions, as well as inadvertent subcutaneous (SC) inoculation. We hypothesized that the antibody and T cell-mediated immune responses (T-CMI) of elderly adults to a vaccine intended for intramuscular (IM) administration would be attenuated when deposited into SC fat, independent of confounding conditions. RESULTS: Fifty-two healthy, community dwelling elderly adults (65-82 years), seronegative for HBV, were enrolled in the SENIEUR protocol as a strictly healthy population. These seniors were randomized to receive a licensed alum-adjuvanted recombinant HBV vaccine either SC or IM, with the inoculum site verified by imaging. The response rates, defined as hepatitis B surface antibodies (HBsAb) ≥10 IU/L, were significantly lower in the elderly than in young adults, a group of 12, healthy, 21-34-year-old volunteers. Moreover, elderly participants who received the vaccine IM were significantly more likely to be responders than those immunized SC (54% versus 16%, p = 0.008). The low seroconversion rate in the IM group progressively declined with increasing age, and responders had significantly lower HBsAb titers and limited isotype responses. Moreover, T-CMI (proliferation and cytokine production) were significantly reduced in both percentage of responders and intensity of the response for both Th1 and Th2 subsets in the elderly. CONCLUSIONS: Our data demonstrate the blunted immunogenicity of SC inoculation as measured by peak titers and response rates. Further, the qualitative and quantitative deficits in B- and T-CMI responses to primary alum adjuvanted protein antigens persisted even in strictly healthy elderly populations with verified IM placement compared to younger populations. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov, NCT04162223. Registered 14 November 2019. Retrospectively registered.

Oral typhoid vaccine Ty21a elicits antigen-specific resident memory CD4+ T cells in the human terminal ileum lamina propria and epithelial compartments.

BACKGROUND: Salmonella enterica serovar Typhi (S. Typhi) is a highly invasive bacterium that infects the human intestinal mucosa and causes ~ 11.9-20.6 million infections and ~ 130,000-223,000 deaths annually worldwide. Oral typhoid vaccine Ty21a confers a moderate level of long-lived protection (5-7 years) in the field. New and improved vaccines against enteric pathogens are needed but their development is hindered by a lack of the immunological correlates of protection especially at the site of infection. Tissue resident memory T (TRM) cells provide immediate adaptive effector immune responsiveness at the infection site. However, the mechanism(s) by which S. Typhi induces TRM in the intestinal mucosa are unknown. Here, we focus on the induction of S. Typhi-specific CD4+TRM subsets by Ty21a in the human terminal ileum lamina propria and epithelial compartments. METHODS: Terminal ileum biopsies were obtained from consenting volunteers undergoing routine colonoscopy who were either immunized orally with 4 doses of Ty21a or not. Isolated lamina propria mononuclear cells (LPMC) and intraepithelial lymphocytes (IEL) CD4+TRM immune responses were determined using either S. Typhi-infected or non-infected autologous EBV-B cell lines as stimulator cells. T-CMI was assessed by the production of 4 cytokines [interferon (IFN)γ, interleukin (IL)-2, IL-17A and tumor necrosis factor (TNF)α] in 36 volunteers (18 vaccinees and 18 controls volunteers). RESULTS: Although the frequencies of LPMC CD103+ CD4+TRM were significant decreased, both CD103+ and CD103- CD4+TRM subsets spontaneously produced significantly higher levels of cytokines (IFNγ and IL-17A) following Ty21a-immunization. Importantly, we observed significant increases in S. Typhi-specific LPMC CD103+ CD4+TRM (IFNγ and IL-17A) and CD103- CD4+TRM (IL-2 and IL-17A) responses following Ty21a-immunization. Further, differences in S. Typhi-specific responses between these two CD4+TRM subsets were observed following multifunctional analysis. In addition, we determined the effect of Ty21a-immunization on IEL and observed significant changes in the frequencies of IEL CD103+ (decrease) and CD103- CD4+TRM (increase) following immunization. Finally, we observed that IEL CD103- CD4+TRM, but not CD103+ CD4+TRM, produced increased cytokines (IFNγ, TNFα and IL-17A) to S. Typhi-specific stimulation following Ty21a-immunization. CONCLUSIONS: Oral Ty21a-immunization elicits distinct compartment specific immune responses in CD4+TRM (CD103+ and CD103-) subsets. This study provides novel insights in the generation of local vaccine-specific responses. Trial registration This study was approved by the Institutional Review Board and registered on ClinicalTrials.gov (identifier NCT03970304, Registered 29 May 2019-Retrospectively registered, http://www.ClinicalTrials.gov/NCT03970304).

Characteristics of regulatory T-cell populations before and after Ty21a typhoid vaccination in children and adults.

Typhoid fever, caused by the pathogen Salmonella enterica serovar Typhi (S. Typhi), is a serious global health concern. Challenge studies with wild type S. Typhi identified associations between gut-homing regulatory T cells (Treg) and development of typhoid disease. Whether oral live-attenuated Ty21a vaccination induces gut-homing Treg remains unclear. Here, we analyze pediatric and adult Treg pre- and post-Ty21a vaccination in an autologous S. Typhi-antigen presentation model to address this knowledge gap. We show that peripheral memory Treg populations change from childhood to adulthood, but not following Ty21a vaccination. Unsupervised dimensionality reduction with t-distributed stochastic neighbor embedding (tSNE) identifies homing, memory, and functional features which evidence age-associated maturation of multifunctional S. Typhi-responsive Treg, which were not impacted by Ty21a vaccination. These findings improve understanding of pediatric regulatory T cells, while identifying age-related differences in S. Typhi-responsive Treg, which may aid in the development of improved pediatric vaccination strategies against S. Typhi.

Diversity of Salmonella Typhi-responsive CD4 and CD8 T cells before and after Ty21a typhoid vaccination in children and adults.

Typhoid fever is a life-threatening disease caused by the human-restricted pathogen Salmonella enterica serovar Typhi (S. Typhi). The oral live attenuated Ty21a typhoid vaccine protects against this severe disease by eliciting robust, multifunctional cell-mediated immunity (CMI), shown to be associated with protection in wild-type S. Typhi challenge studies. Ty21a induces S. Typhi-responsive CD8+ and CD4+ T cells but little is known about the response to this vaccine in children. To address this important gap in knowledge, we have used mass cytometry to analyze pediatric and adult pre- and post-Ty21a vaccination CMI in an autologous S. Typhi antigen presentation model. Here, using conventional supervised analytical tools, we show adult T cells are more multifunctional at baseline than those obtained from children. Moreover, pediatric and adult T cells respond similarly to Ty21a vaccination, but adult responders remain more multifunctional. The use of the unsupervised dimensionality reduction tool tSNE (t-distributed Stochastic Neighbor Embedding) allowed us to confirm these findings, as well as to identify increases and decreases in well-defined specific CD4+ and CD8+ T-cell populations that were not possible to uncover using the conventional gating strategies. These findings evidenced age-associated maturation of multifunctional S. Typhi-responsive T-cell populations, including those which we have previously shown to be associated with protection from, and/or delayed onset of, typhoid disease. These findings are likely to play an important role in improving pediatric vaccination strategies against S. Typhi and other enteric pathogens.

Attenuated Oral Typhoid Vaccine Ty21a Elicits Lamina Propria and Intra-Epithelial Lymphocyte Tissue-Resident Effector Memory CD8 T Responses in the Human Terminal Ileum.

Tissue-resident memory T cells (TRM) are newly defined memory T cells (TM) distinct from circulating TM subsets which have the potential to mount rapid protective immune responses at the site of infection. However, very limited information is available regarding the role and contribution of TRM in vaccine-mediated immune responses in humans at the site of infection. Here, we studied the role and contribution of tissue resident memory T cells (TRM) located in the terminal ileum (TI) (favored site of infection for S. Typhi) following oral Ty21a immunization in humans. We examined TI-lamina propria mononuclear cells (LPMC) and intra-epithelial lymphocytes (IEL) CD8+ TRM subsets obtained from healthy volunteers undergoing medically-indicated colonoscopies who were either immunized with Ty21a or unvaccinated. No significant differences in the frequencies of LPMC CD8+ TRM and CD8+CD69+CD103- T cells subsets were observed following Ty21a-immunization. However, LPMC CD8+ TRM exhibited significantly higher levels of cytokines (IFN-γ, IL-17A, and TNF-α) ex-vivo in Ty21a-vaccinated than in unvaccinated volunteers. LPMC CD8+ TRMS. Typhi-specific responses were evaluated using S. Typhi-infected targets and found to produce significantly higher levels of S. Typhi-specific IL-17A. In contrast, LPMC CD8+CD69+CD103- T cells produced significantly increased S. Typhi-specific levels of IFN-γ, IL-2, and IL-17A. Finally, we assessed CD8+ TRM in IEL and observed that the frequency of IEL CD8+ TRM is significantly lower following Ty21a immunization. However, ex-vivo IEL CD8+ TRM elicited by Ty21a immunization spontaneously produced significantly higher levels of cytokines (IFN-γ, IL-17A, IL-2, and TNF-α). This study provides the first demonstration of the effect of oral Ty21a vaccination on CD8+ TRM subsets (spontaneous and S. Typhi-specific) responses in the LPMC and IEL compartment of the human terminal ileum mucosa, contributing novel information to our understanding of the generation of mucosal immune responses following oral Ty21a-immunization.

Age-Associated Heterogeneity of Ty21a-Induced T Cell Responses to HLA-E Restricted Salmonella Typhi Antigen Presentation.

Human-restricted Salmonella enterica serovar Typhi (S. Typhi) is the causative agent of typhoid fever-a life-threatening disease of great global health significance, particularly in the developing world. Ty21a is an oral live-attenuated vaccine that protects against the development of typhoid disease in part by inducing robust T cell responses, among which multifunctional CD8+ cytotoxic T lymphocytes (CTL) play an important role. Following Ty21a vaccination, a significant component of adult CTL have shown to be targeted to S. Typhi antigen presented by the conserved major histocompatibility complex (MHC) class Ib molecule, human leukocyte antigen-E (HLA-E). S. Typhi challenge studies have shown that baseline, multifunctional HLA-E responsive T cells are associated with protection from, and delayed onset of, typhoid disease. However, despite the overwhelming burden of typhoid fever in school-aged children, and due to limited availability of pediatric samples, incomplete information is available regarding these important HLA-E-restricted responses in children, even though studies have shown that younger children may be less likely to develop protective cell mediated immune (CMI) responses than adults following vaccination. To address this gap, we have studied this phenomenon in depth by using mass cytometry to analyze pediatric and adult T cell responses to HLA-E-restricted S. Typhi antigen presentation, before and after Ty21a vaccination. Herein, we show variable responses in all age strata following vaccination among T effector memory (TEM) and T effector memory CD45RA+ (TEMRA) cells based on conventional gating analysis. However, by utilizing the dimensionality reduction tool tSNE (t-distributed Stochastic Neighbor Embedding), we are able to identify diverse, highly multifunctional gut-homing- TEM and TEMRA clusters of cells which are more abundant in adult and older pediatric participants than in younger children. These findings highlight a potential age-associated maturation of otherwise conserved HLA-E restricted T cell responses. Such insights, coupled with the marked importance of multifunctional T cell responses to combat infection, may better inform future pediatric vaccination strategies against S. Typhi and other infectious diseases.

Effect of the live oral attenuated typhoid vaccine, Ty21a, on systemic and terminal ileum mucosal CD4+ T memory responses in humans.

Our current understanding of CD4+ T-cell-mediated immunity (CMI) elicited by the oral live attenuated typhoid vaccine Ty21a is primarily derived from studies using peripheral blood. Very limited data are available in humans regarding mucosal immunity (especially CD4+ T) at the site of infection (e.g. terminal ileum; TI). Here using multiparametric flow cytometry, we examined the effect of Ty21a immunization on TI-lamina propria mononuclear cells (LPMC) and peripheral blood CD4+ T memory (TM) subsets in volunteers undergoing routine colonoscopy. Interestingly, we observed significant increases in the frequencies of LPMC CD4+ T cells following Ty21a immunization, restricted to the T effector/memory (TEM)-CD45RA+ (TEMRA) subset. Importantly, Ty21a immunization elicited Salmonella Typhi-responsive LPMC CD4+ T cells in all major TM subsets [interferon (IFN)γ and interleukin (IL)-17A in TEM; IFNγ and macrophage inflammatory protein (MIP)1ß in T central/memory (TCM); and IL-2 in TEMRA]. Subsequently, we analyzed LPMC S. Typhi-responsive CD4+ T cells in depth for multifunctional (MF) effectors. We found that LPMC CD4+ TEM responses were mostly MF, except for those cells exhibiting the characteristics associated with IL-17A responses. Finally, we compared mucosal to systemic responses and observed that LPMC CD4+S. Typhi-specific responses were unique and distinct from their systemic counterparts. This study provides the first demonstration of S. Typhi-specific CD4+ TM responses in the human TI mucosa and provides valuable information about the generation of mucosal immune responses following oral Ty21a immunization.

Differences Between Pediatric and Adult T Cell Responses to In Vitro Staphylococcal Enterotoxin B Stimulation.

Toxic shock syndrome (TSS) is capable of inducing life-threatening fever, rash, and systemic organ failure, though the specific mechanisms behind these symptoms remain poorly understood. Staphylococcal enterotoxin B (SEB) and other superantigens have shown to be important factors in TSS, capable of promoting cross-linking between T cell receptors and major histocompatibility complexes which results in overwhelming T cell activation, proliferation, and cytokine production. The resulting proinflammatory cytokine cascade, often referred to as the "cytokine storm," seems to be critical to the development of disease. Interestingly, clinical studies have shown that children exhibit less severe TSS-associated morbidity than adults, though the mechanism behind this phenomenon has not been addressed. Indeed, despite the fact that most novel antigen exposure occurs early in life, be it from environmentally acquired pathogens or routine vaccination, normal pediatric T cell immune functions remain critically underexplored. This is largely due to difficulty in obtaining enough samples to explore more than a narrow sliver of the cell-mediated immune compartment. To address this limitation, we optimized a T effector (Teff)/circulating T follicular helper (cTFH) cell mass cytometry panel which allowed us to analyze a wide array of T cell populations and effector functions following in vitro SEB stimulation. We show that T cell activation-as measured by CD69 expression-following SEB stimulation is lower in pediatric participants, increasing throughout childhood, and reaching adult levels by around 15 years old. Further, while individual CD4+ effector memory T cell (TEM) effector molecules show limited age-associated differences following SEB stimulation, multifunctional CD4+ TEM are shown to positively correlate with increasing age through adolescence. Individual CD8+ TEM effectors and multifunctional phenotypes also show very strong age-associated increases following SEB stimulation. SEB stimulation has little impact on cTFH activation or functional cellular markers, regardless of age. These results, coupled with the fact that a robust proinflammatory cytokine response seems critical to developing severe TSS, suggest a possible connection between the significantly reduced T cell activation and multifunctional populations following in vitro SEB stimulation in our pediatric participants and clinical observations relating to reduced TSS mortality in children.