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The current pneumococcal capsular polysaccharide (PPS) conjugate vaccine (PCV13) is less effective againstStreptococcus pneumoniaeserotype 3 (ST3), which remains a major cause of pneumococcal disease and mortality. Therefore, dissecting structure-function relationships of human PPS3 antibodies may reveal characteristics of protective antibodies. Using flow cytometry, we isolated PPS3-binding memory B cells from pneumococcal vaccine recipients and generated seven human PPS3-specific monoclonal antibodies (humAbs). Five humAbs displayed ST3 opsonophagocytic activity, four induced ST3 agglutinationin vitro, and four mediated both activities. For two humAbs, C10 and C27, that used the same variable heavy (VH) and light (VL) chain domains (VH3-9*01/VL2-14*03), C10 had fewer VLsomatic mutations, higher PPS3 affinity, more ST3 opsonophagocytic and agglutinating activity, whilst both humAbs altered ST3 gene expressionin vitro. After VLswaps, C10VH/C27VLexhibited reduced ST3 binding and agglutination, but C27VH/C10VLbinding was unchanged. In C57Bl/6 mice, C10 and C27 reduced nasopharyngeal colonization with ST3 A66 and a clinical strain, B2, and prolonged survival following lethal A66 intraperitoneal infection, but only C10 protected against lethal intranasal infection with the clinical strain. Our findings, associate efficacy of PPS3-specific humAbs with ST3 agglutination and opsonophagocytic activity and reveal an unexpected role for the VLin functional activityin vitroandin vivo. These findings also provide insights that may inform antibody-based therapy and identification of surrogates of vaccine efficacy against ST3.IMPORTANCEDespite the global success of pneumococcal conjugate vaccination, serotype 3 (ST3) pneumococcus remains a leading cause of morbidity and mortality. In comparison to other vaccine-included serotypes, the ST3 pneumococcal capsular polysaccharide (PPS3) induces a weaker opsonophagocytic response, which is considered a correlate of vaccine efficacy. Previous studies of mouse PPS3 monoclonal antibodies identified ST3 agglutination as a correlate of reduced ST3 nasopharyngeal colonization in mice, however neither the agglutinating ability of human vaccine-elicited PPS3 antibodies nor their ability to prevent experimental murine nasopharyngeal colonization has been studied. We generated and analysed the functional andin vivoefficacy of human vaccine-elicited PPS3 monoclonal antibodies and found that ST3 agglutination associated with antibody affinity, protectionin vivo, and limited somatic mutations in the light chain variable region. These findings provide new insights that may inform the development of antibody-based therapies and next generation vaccines for ST3.

Kara et al. show that ACKR4 regulates early activated B cell differentiation fate. ACKR4 limits the early proliferation of activated B cells by restricting their initial access to the splenic interfollicular zone., Activated B cells can initially differentiate into three functionally distinct fates—early plasmablasts (PBs), germinal center (GC) B cells, or early memory B cells—by mechanisms that remain poorly understood. Here, we identify atypical chemokine receptor 4 (ACKR4), a decoy receptor that binds and degrades CCR7 ligands CCL19/CCL21, as a regulator of early activated B cell differentiation. By restricting initial access to splenic interfollicular zones (IFZs), ACKR4 limits the early proliferation of activated B cells, reducing the numbers available for subsequent differentiation. Consequently, ACKR4 deficiency enhanced early PB and GC B cell responses in a CCL19/CCL21-dependent and B cell–intrinsic manner. Conversely, aberrant localization of ACKR4-deficient activated B cells to the IFZ was associated with their preferential commitment to the early PB linage. Our results reveal a regulatory mechanism of B cell trafficking via an atypical chemokine receptor that shapes activated B cell fate.

Gamma-irradiation, particularly an irradiation dose of 50 kGy, has been utilised widely to sterilise highly pathogenic agents such as Ebola, Marburg Virus, and Avian Influenza H5N1. We have reported previously that intranasal vaccination with a gamma-irradiated Influenza A virus vaccine (γ-Flu) results in cross-protective immunity. Considering the possible inclusion of highly pathogenic Influenza strains in future clinical development of γ-Flu, an irradiation dose of 50 kGy may be used to enhance vaccine safety beyond the internationally accepted Sterility Assurance Level (SAL). Thus, we investigated the effect of irradiation conditions, including high irradiation doses, on the immunogenicity of γ-Flu. Our data confirm that irradiation at low temperatures (using dry-ice) is associated with reduced damage to viral structure compared with irradiation at room temperature. In addition, a single intranasal vaccination with γ-Flu irradiated on dry-ice with either 25 or 50 kGy induced seroconversion and provided complete protection against lethal Influenza A challenge. Considering that low temperature is expected to reduce the protein damage associated with exposure to high irradiation doses, we titrated the vaccine dose to verify the efficacy of 50 kGy γ-Flu. Our data demonstrate that exposure to 50 kGy on dry-ice is associated with limited effect on vaccine immunogenicity, apparent only when using very low vaccine doses. Overall, our data highlight the immunogenicity of influenza virus irradiated at 50 kGy for induction of high titre antibody and cytotoxic T-cell responses. This suggests these conditions are suitable for development of γ-Flu vaccines based on highly pathogenic Influenza A viruses.

Klebsiella pneumoniae ST258 is a globally distributed multi-drug resistant pathogen responsible for severe invasive infections. In this study, the different virulence potential of K. pneumoniae ST258 isolates in endotoxin susceptible versus resistant animal models was shown. Furthermore, ST258 clinical isolates were found highly sensitive to the bactericidal effect of naive animal and human serum. These observations imply that LPS, released from the rapidly lysed bacteria, may contribute to the high mortality associated with ST258 bacteremia cases. A humanized version (mAb A1102) of a previously described murine mAb specific for the conserved LPS O-antigen, was tested for endotoxin neutralization. A1102 was able to neutralize TLR-4 activation by ST258-derived LPS in vitro with an efficacy exceeding that of polymyxin B by 3 orders of magnitude. Passive immunization with A1102 afforded a significant level of protection in a galactosamine-sensitized mouse model of endotoxemia, induced by ST258-derived LPS, or upon challenge with live bacteria. Efficacy was retained using an aglycosylated IgG, as well as upon complement depletion, suggesting that Fc-independent endotoxin neutralization may be the main protective mechanism in this model, in spite of the complement-dependent bactericidal and opsonic activities additionally observed for A1102 in vitro. Furthermore, rabbits that are naturally highly susceptible to endotoxin, were also significantly protected by low doses of A1102 when challenged with an ST258 strain. Given this unique mode of action and the high protective efficacy of this mAb, passive immunization, as prophylactic or adjunct therapeutic approach for the treatment of infections caused by ST258 isolates should be considered.

Background Streptococcus pneumoniae is the main bacterial cause of pneumonia in the United States and globally. Although pneumococcal conjugate vaccines are highly effective against invasive pneumococcal disease, they are less effective against pneumonia, particularly in the elderly and those with immune deficiency. Given the additional challenge of antibiotic resistance, immunotherapy holds promise for treatment of pneumococcal pneumonia. The current PCV13 vaccine is less effective against serotype (ST) 3, which carries a higher risk of mortality than other vaccine-included STs. Our group has previously identified murine monoclonal antibodies (mAb) to ST3 capsular polysaccharide (PPS3) that are protective in experimental models of sepsis and pneumonia. The aim of the present study is to isolate and develop PPS3-specific human monoclonal antibodies (humAbs) as adjunctive immunotherapy for pneumonia. Methods We sorted individual PPS3-specific memory B cells from PBMCs isolated on days 0 and 7 post-vaccination from pneumococcal polysaccharide (PPS)-based vaccine (Pneumovax or Prevnar13) recipients using fluorescently labeled PPS3. Immunoglobulin heavy (Igh) and light (Igl) chains were sequenced, cloned into IgG1 and κ or λ vectors, and expressed in HEK-293 cells. PPS3 specificity was confirmed using ELISA. Results Here, we report the first 7 PPS3-specific humAbs isolated: 5 used lambda light chains and two used kappa light chains. Six of these humAbs used variable heavy 3 (VH3) Igh gene elements. Kappa humAbs used VH3-30 or VH3-7, whilst lambda humAbs used VH3-9, VH3-72 or VH3-23. Sequence analysis revealed somatic mutations in complementary determining as well as framework regions. Initial studies show that some humAbs agglutinate ST3 in vitro. Structure-function relationship studies are ongoing to identify specific determinants of PPS3 binding and biological efficacy against ST3 in vitro and in vivo. Conclusion The results of this study provide further understanding of the biology of PPS3 antibodies and may facilitate design of adjunctive immunotherapy to treat and prevent ST3 disease. Disclosures All authors: No reported disclosures.

Interleukin 17-producing γδ T (γδT17) cells have unconventional trafficking characteristics, residing in mucocutaneous tissues but also homing into inflamed tissues via circulation. Despite being fundamental to γδT17-driven early protective immunity and exacerbation of autoimmunity and cancer, migratory cues controlling γδT17 cell positioning in barrier tissues and recruitment to inflammatory sites are still unclear. Here we show that γδT17 cells constitutively express chemokine receptors CCR6 and CCR2. While CCR6 recruits resting γδT17 cells to the dermis, CCR2 drives rapid γδT17 cell recruitment to inflamed tissues during autoimmunity, cancer and infection. Downregulation of CCR6 by IRF4 and BATF upon γδT17 activation is required for optimal recruitment of γδT17 cells to inflamed tissue by preventing their sequestration into uninflamed dermis. These findings establish a lymphocyte trafficking model whereby a hierarchy of homing signals is prioritized by dynamic receptor expression to drive both tissue surveillance and rapid recruitment of γδT17 cells to inflammatory lesions., IL-17-producing γδ T (γδT17) cells position in barrier tissues but also home to inflammatory sites. How this trafficking is regulated is unclear. Here the authors show that the dynamic expression of chemokine receptors CCR2 and CCR6 differentiates γδT17 cell trafficking patterns at homeostasis and in inflammatory scenarios.

Generating a pneumococcal vaccine that is serotype independent and cost effective remains a global challenge. γ-Irradiation has been used widely to sterilize biological products. It can also be utilized as an inactivation technique to generate whole-cell bacterial and viral vaccines with minimal impact on pathogen structure and antigenic determinants. In the present study, we utilized γ-irradiation to inactivate an un-encapsulated Streptococcus pneumoniae strain Rx1 with an unmarked deletion of the autolysin gene lytA and with the pneumolysin gene ply replaced with an allele encoding a non-toxic pneumolysoid (PdT) (designated γ-PN vaccine). Intranasal vaccination of C57BL/6 mice with γ-PN was shown to elicit serotype-independent protection in lethal challenge models of pneumococcal pneumonia and sepsis. Vaccine efficacy was shown to be reliant on B-cells and interleukin (IL)-17A responses. Interestingly, immunization promoted IL-17 production by innate cells not T helper 17 (Th17) cells. These data are the first to report the development of a non-adjuvanted intranasal γ-irradiated pneumococcal vaccine that generates effective serotype-independent protection, which is mediated by both humoral and innate IL-17 responses. Refereed/Peer-reviewed

During acute viral infections, innate immunity provides essential protective measures to minimize virus dissemination and regulate adaptive immunity. This helps to successfully eliminate the pathogen and establish long-term memory. Here, we investigated the effect of vaccine-induced antibody responses on the induction of IFN-I responses and the associated lymphocyte activation using influenza A virus vaccination and challenge models. Mice were vaccinated with gamma-irradiated influenza A virus (γ-FLU) and challenged three weeks later with live virus. Our data show a significant reduction in IFN-I responses and lymphocyte activation following a homotypic virus challenge. We confirmed the role of vaccine-induced antibody responses in the observed impairment of IFN-I and the associated lymphocyte activation using adoptive transfer of immune sera and the administration of sera-treated viruses prior to challenge. Overall, we addressed a fundamental concept in immunology and provided experimental data illustrating the inhibition of IFN-I responses in vaccinated animals upon a homotypic virus challenge.