Mucosal IgA Antibody Responses Research Articles

Measles Virus-Based Vaccine Expressing Membrane-Anchored Spike of SARS-CoV-2 Inducing Efficacious Systemic and Mucosal Humoral Immunity in Hamsters.

As SARS-CoV-2 continues to evolve and COVID-19 cases rapidly increase among children and adults, there is an urgent need for a safe and effective vaccine that can elicit systemic and mucosal humoral immunity to limit the emergence of new variants. Using the Chinese Hu191 measles virus (MeV-hu191) vaccine strain as a backbone, we developed MeV chimeras stably expressing the prefusion forms of either membrane-anchored, full-length spike (rMeV-preFS), or its soluble secreted spike trimers with the help of the SP-D trimerization tag (rMeV-S+SPD) of SARS-CoV-2 Omicron BA.2. The two vaccine candidates were administrated in golden Syrian hamsters through the intranasal or subcutaneous routes to determine the optimal immunization route for challenge. The intranasal delivery of rMeV-S+SPD induced a more robust mucosal IgA antibody response than the subcutaneous route. The mucosal IgA antibody induced by rMeV-preFS through the intranasal routine was slightly higher than the subcutaneous route, but there was no significant difference. The rMeV-preFS vaccine stimulated higher mucosal IgA than the rMeV-S+SPD vaccine through intranasal or subcutaneous administration. In hamsters, intranasal administration of the rMeV-preFS vaccine elicited high levels of NAbs, protecting against the SARS-CoV-2 Omicron BA.2 variant challenge by reducing virus loads and diminishing pathological changes in vaccinated animals. Encouragingly, sera collected from the rMeV-preFS group consistently showed robust and significantly high neutralizing titers against the latest variant XBB.1.16. These data suggest that rMeV-preFS is a highly promising COVID-19 candidate vaccine that has great potential to be developed into bivalent vaccines (MeV/SARS-CoV-2).

Glycosylated Delta-receptor-binding domain mucosal vaccine elicits broadly neutralizing antibodies with protection against SARS-CoV-2 challenge

Mucosal COVID-19 vaccines are needed to block SARS-CoV-2 infection at the mucosal site. Intranasal delivery of a glycosylated Delta variant receptor-binding domain (Delta-RBD) mucosal vaccine elicited potent and balanced systemic antibody titers comparable to those induced by the intramuscular injection of the same vaccine or Omicron-S subunit vaccine, as well as high mucosal IgA antibody responses. It elicited broadly neutralizing antibodies against the original SARS-CoV-2 strain, Delta and Omicron BA1/BA2 variants, completely protecting transgenic mice from lethal challenge with a Delta variant, including complete absence of weight loss. Of note, intramuscular priming with the Omicron-S protein followed by intranasal boosting with the Delta-RBD protein improved the vaccine's ability to generate broad-spectrum neutralizing antibodies against recent BA5 and XBB Omicron variants. Overall, this vaccine has the potential to prevent the SARS-CoV-2 infection of the respiratory mucosa, while the i.m. priming and i.n. boosting vaccination strategy may offer protection against known and emerging SARS-CoV-2 variants.

Chicken dendritic cell-targeting nanobodies mediated improved protective effects against H9N2 influenza virus challenge in a homologous sequential immunization study

Global poultry production is still severely affected by H9N2 avian influenza virus (AIV), and the development of a novel universal AIV vaccine is still urgently needed. Neuraminidase (NA) has recently been shown to be an efficient conserved protective antigen.In this study, we fused the extracellular region of the NA gene with a ferritin cassette (pYL281), which resulted in self-assembled 24-mer nanoparticles with the NA protein displayed outside the nanoparticles. In addition, a chicken dendritic cell-targeting nanobody-phage74 was also inserted ahead of the NA protein to yield pYL294. Incubation with chicken bone marrow-derived dendritic cells (chBMDCs) showed that the DC-targeting nanoparticles purified from the pYL294 strain significantly increased the maturation of chBMDCs, as shown by increased levels of CCL5, CCR7, CD83 and CD86 compared with nontargeting proteins. Then, a chicken study was performed using Salmonella oral administration together with intranasal boost with purified proteins. Compared with the other groups, oral immunization with Salmonella harboring pYL294 followed by intranasal boost with purified DC-targeting nanoparticles dramatically increased the humoral IgY and mucosal IgA antibody response, as well as increased the cellular immune response, as shown by elevated splenic lymphocyte proliferation and intracellular mRNA levels of IL-4 and IFN-γ. Finally, sequential immunization with DC-targeting nanoparticles showed increased protection against G57 subtype H9N2 virus challenge compared with other groups, as shown by significantly decreased virus RNA copy numbers in oropharyngeal washes (Days 3, 5 and 7 post challenge) and cloacal washes (Day 7), significantly decreased lung virus titers on Day 5 post challenge and increased body weight gains during the challenge.

Targeting MAIT cells as a cellular adjuvant for humoral immunity: a new player in a very old game.

In this article, I discuss recent work by Pankhurst etal. They found that MAIT cells can serve as a cellular adjuvant to boost immunity to a protein adjuvant. Intranasal co-administration of protein antigen with a strong MAIT cell ligand results in the the production of mucosal IgA and IgG antibody responses. This process is driven by MAIT cell-mediated maturation of migratory dendritic cells.

Application of M-cell targeting ligand Co4B enhances Ag-specific mucosal and systemic immunity and protection against MERS-CoV infection after intranasal immunization

Abstract Middle East respiratory syndrome is associated with severe pneumonia and poses a global public health threat. Authorized vaccines against the causative agent of MERS-CoV are urgently needed. In order to optimize the efficacy of recombinant vaccines, adjuvants should be required. Therefore, we previously identified a ligand Co4B as an adjuvant to enhance Ag delivery to the nasal mucosa immune compartment and promote Ag-specific mucosal and systemic immune responses via intranasal administration against A. pleuropneumoniae infection in a murine model. Based on the pathogenesis of MERS-CoV which infects host following respiratory route; thus, we generated a recombinant protein conjugated with Co4B to the MERS-CoV spike protein receptor-binding domain (S-RBD) to develop mucosal subunit vaccine candidate. Ag-specific mucosal IgA and systemic IgG antibody responses, together with virus-neutralizing capacities, were highly elicited in C57BL/6 and human dipeptidyl peptidase 4-transgenic (hDPP4-Tg) mice intranasally immunized with Co4B-conjugated S-RBD compared to those immunized with originally S-RBD. Ag-specific T cell-mediated immunity was also induced in the spleen and lungs of mice intranasally immunized with Co4B-conjugated S-RBD. Intranasal immunization of hDPP4-Tg mice with Co4B-conjugated S-RBD prominently reduced immune cell infiltration into the tissues of virus-challenged mice. Finally, Co4B-conjugated S-RBD-immunized mice demonstrated were improved protection against infection, more likely to survive, and exhibited less body weight loss. Collectively, our results suggest that Co4B-conjugated S-RBD could be used as an improved subunit vaccine candidate against MERS-CoV infection. This work was supported by the Ministry of Science and ICT No. 2020K1A4A7A02095058 and the NRF funded by the Ministry of Education No. 2019R1I1A3A01062224. Ms. Y. L. Yang and Ms. B.-H. Cho were supported by the BK21 FOUR program of the Department of Bioactive Material Sciences.

Safety, Immunogenicity, and Protective Efficacy of an H5N1 Chimeric Cold-Adapted Attenuated Virus Vaccine in a Mouse Model

H5N1 influenza virus is a threat to public health worldwide. The virus can cause severe morbidity and mortality in humans. We constructed an H5N1 influenza candidate virus vaccine from the A/chicken/Guizhou/1153/2016 strain that was recommended by the World Health Organization. In this study, we designed an H5N1 chimeric influenza A/B vaccine based on a cold-adapted (ca) influenza B virus B/Vienna/1/99 backbone. We modified the ectodomain of H5N1 hemagglutinin (HA) protein, while retaining the packaging signals of influenza B virus, and then rescued a chimeric cold-adapted H5N1 candidate influenza vaccine through a reverse genetic system. The chimeric H5N1 vaccine replicated well in eggs and the Madin-Darby Canine Kidney cells. It maintained a temperature-sensitive and cold-adapted phenotype. The H5N1 vaccine was attenuated in mice. Hemagglutination inhibition (HAI) antibodies, micro-neutralizing (MN) antibodies, and IgG antibodies were induced in immunized mice, and the mucosal IgA antibody responses were detected in their lung lavage fluids. The IFN-γ-secretion and IL-4-secretion by the mouse splenocytes were induced after stimulation with the specific H5N1 HA protein. The chimeric H5N1 candidate vaccine protected mice against lethal challenge with a wild-type highly pathogenic avian H5N1 influenza virus. The chimeric H5 candidate vaccine is thus a potentially safe, attenuated, and reassortment-incompetent vaccine with circulating A viruses.

Comparison of the mucosal adjuvanticities of two Toll-like receptor ligands for recombinant adenovirus-delivered African swine fever virus fusion antigens

The mucosal immunity plays an important role against African swine fever virus (ASFV) infection and the efficacy of mucosal vaccination is highly dependent on the adjuvant. However, the mucosal adjuvant for ASFV vaccination is poorly studied. Toll-like receptor (TLR) ligands such as the FlaB flagellin from Vibrio vulnificus and the heat shock protein 70 from Mycobacterium tuberculosis (mHsp70) hold a great promise as novel vaccine adjuvant. However, the mucosal adjuvanticities of such TLR ligands have not been studied in pigs. In this study, three recombinant Adenovirus (rAd) vectors, namely rAd-F1, rAd-FlaB-F1 and rAd-F1-Hsp70, were constructed by fusing the FlaB or mHsp70 to ASFV CD2v-p30-p54 fusion antigen. Western blotting showed that the three fusion proteins expressed in rAd-infected cells reacted positively with ASFV antibodies. After intranasal immunization of pigs with the three rAd vectors, the antigen-specific IgG antibodies were detectable from day 7 after primary immunization, which were significantly boosted by the secondary immunization. Strong Th1/Th2 cytokine responses were detected in the peripheral blood mononuclear cells. Compared to immunization with the control rAd-F1, significantly higher levels of the antigen-specific IgA antibodies were detected in the nasal fluids, tracheal washes and lung lavages.11of rAd-FlaB-F1- or rAd-F1-Hsp70-immunized pigs. Compared to immunization with rAd-Flab-F1, immunization with rAd-F1-Hsp70 induced significantly stronger mucosal IgA antibody response. Cytokine detection of the pig lung lavages showed that the elevated IgA antibody responses were correlated mainly with IL-4, IL-10 and IFN-α, which were confirmed by the significantly increased antigen-recall cytokine expression in the porcine alveolar macrophages. These data suggest that mHsp70 has potent mucosal adjuvanticity in pigs, and the fusion rAd vector can be used for ASFV mucosal vaccine development.

Hydroxypropyltrimethyl ammonium chloride chitosan-based hydrogel as the split H5N1 mucosal adjuvant: Structure-activity relationship

In this study, 2-hydroxypropyltrimethyl ammonium chloride chitosan (HTCC)-based hydrogel was devised as a mucosal adjuvant for H5N1 vaccine. Aimed to investigate the structure activity relationship between HTCC hydrogel and immune response, we prepared a series of HTCC hydrogel with defined quaternization degrees (DQs, 0%, 21%, 41%, 60%, 80%). Results suggested that with DQ increasing, the positive charge and gelation time of HTCC hydrogel increased but the viscosity decreased. We applied in vivo imaging system and found that the moderate DQ 41% prolonged antigen residence time in nasal cavity, resulting in the most potent systemic responses (IgG, IgG1, IgG2a, HI). While, the lowest DQ 0% produced the best mucosal IgA antibody responses, most likely due to the closer contact with mucosa. Furthermore, the influence of animal gender was also discussed. These data add to the growing understanding of the relationship between physicochemical features of chitosan-based hydrogel and how they influence the immune responses.

VA-MENGOC-BC Vaccination Induces Serum and Mucosal Anti Neisseria gonorrhoeae Immune Responses and Reduces the Incidence of Gonorrhea.

Overall, there are over 30 different sexually transmitted infections with Neisseria gonorrhoeae being the third most frequent with a reported 78 million cases per year. Gonococcal infection causes genital inflammation, which can be a risk factor for others sexually transmitted infections, particularly human immunodeficiency virus. Gonorrhea is a treatable disease, but recently an increase in antibiotic resistance has been of concern. There are currently no vaccines available. However, parenteral vaccination with anti N. meningitidis serogroup B vaccine has been reported to decrease the incidence of gonococcal burden in New Zealand and in Cuba despite the fact that parenteral vaccination is not deemed to induce mucosal IgA. Here we explore possible mechanisms of protection against gonococcal infection through parenteral meningococcal B vaccination. Ninety-two serum, saliva and oropharyngeal swabs samples of young adults (healthy and Neisseria carriers) of the internal higher school were obtained. They have been vaccinated with VA-MENGOC-BC (MBV) during their infancy and boosted with a third dose during this study. Serum and saliva samples were analyzed by ELISA and Western blot to measured IgG and IgA antibodies against N. meningitidis and N. gonorrhoeae antigens. N. meningitidis carriers were determined by standard microbiologic test. In addition, we reviewed epidemiologic data for N. meningitidis and N. gonorrhoeae infections in Cuba. Epidemiologic data show the influence of MBV over gonorrhea incidence suggesting to be dependent of sexual arrival age of vaccines but not over syphilis. Laboratorial data permit the detection of 70 and 22 noncarriers and carriers of N. meningitidis, respectively. Serum anti-MBV antigens (PL) responses were boosted by a third dose and were independent of carriage stages, but saliva anti-PL IgA responses were only present and were significant induced in carriers subjects. Carriers boosted with a third dose of MBV induced similar antigonococcal and -PL saliva IgA and serum IgG responses; meanwhile, serum antigonococcal IgG was significantly lower. In saliva, at least 2 gonococcal antigens were identified by Western blot. Finally, gonococcal-specific mucosal IgA antibody responses, in addition to the serum IgG antibodies, might contributed to the reduction of the incidence of N. gonorrhoeae. We hypothesize that this might have contributed to the observed reductions of the incidence of N. gonorrhoeae. These results suggest a mechanism for the influence of a Proteoliposome-based meningococcal BC vaccine on gonococcal incidence.

Systemic and intestinal porcine epidemic diarrhea virus-specific antibody response and distribution of antibody-secreting cells in experimentally infected conventional pigs

Porcine epidemic diarrhea (PED) is a coronavirus disease characterized by the rapid spread of severe diarrhea among pigs. PED virus (PEDV) infects and replicates mainly in the epithelial cells of the duodenum, jejunum, ileum and colon. Serum or mucosal IgA antibody levels have been used to predict both vaccine efficacy and the level of protective immunity to enteric infectious diseases in individuals or herds. Details of the B-cell immune response upon PEDV infection, such as the systemic and mucosal PEDV IgA antibody response, the distribution of IgA antibody-secreting cells (ASCs), and their role in virus clearance are not yet clear. In this experimental infection study, we observed similar fluctuations in PEDV IgA antibody levels in serum and intestinal contents of the upper and lower jejunum and ileum, but not fecal samples, over the 4-week experimental course. ASCs that actively secrete PEDV IgA antibody without in vitro stimulation were distributed mainly in the upper jejunum, whereas memory B cells that showed enhanced PEDV IgA antibody production upon in vitro stimulation were observed in mesenteric lymph nodes and the ileum. Our findings will contribute to the development of effective vaccines and diagnostic methods for PEDV.

Induction of antibody responses in mice immunized intranasally with Type I interferon as adjuvant and synergistic effect of chitosan.

Type I IFNs are a range of host-derived molecules with adjuvant potential; they have been used for many years in the treatment of cancer and viral hepatitis. Therefore, the safety of IFNs for human use has been established. In this study, we evaluated the mucosal adjuvanticity of IFN-β administered intranasally to mice with diphtheria toxoid, and suggested a method to improve its adjuvanticity. When IFN-β alone was used as a mucosal adjuvant, no clear results were obtained. However, simultaneous administration of IFN-β and chitosan resulted in an enhancement of the specific serum immunoglobulin G (IgG) and IgA antibody responses, the mucosal IgA antibody response, and antitoxin titers. Furthermore, the intranasal administration of IFN-α alone resulted in a greater increase in antibody titer than IFN-β, and a synergistic effect with chitosan was also observed. These findings suggest that intranasal administration of chitosan and Type I IFNs may display an effective synergistic mucosal adjuvant activity.

Enhanced Oral Vaccine Efficacy of Polysaccharide-Coated Calcium Phosphate Nanoparticles.

Oral administration of vaccines has been limited due to low immune response compared to parenteral administration. Antigen degradation in the acidic gastrointestinal environment (GI), mucus barriers, and inefficient cellular uptake by immune cells are the major challenges for oral vaccine delivery. To solve these issues, the current study investigates calcium phosphate nanoparticles (CaP NPs) coated with polysaccharides as nanocarriers for oral protein antigen delivery. In this design, the CaP NP core had an optimized antigen encapsulation capacity of 90 mg (BSA-FITC)/g (CaP NPs). The polysaccharides chitosan and alginate were coated onto the CaP NPs to protect the antigens against acidic degradation in the GI environment and enhance the immune response in the small intestine. The antigen release profiles showed that alginate-chitosan-coated CaP NPs prevented antigen release in a simulated gastric fluid (pH 1.2), followed by sustained release in simulated intestinal (pH 6.8) and colonic (pH 7.4) fluids. Cellular uptake and macrophage stimulation data revealed that the chitosan coating enhanced antigen uptake by intestine epithelia cells (Caco-2) and macrophages and improved surface expression of costimulatory molecules on macrophages. In vivo test further demonstrated that oral administration of alginate-chitosan-coated CaP@OVA NPs significantly enhanced the mucosal IgA and serum IgG antibody responses as compared to naked OVA, indicating that the CaP-Chi-Alg nanoparticle can potentially be used as a promising oral vaccine delivery system.

Intranasally administered protein coated chitosan nanoparticles encapsulating influenza H9N2 HA2 and M2e mRNA molecules elicit protective immunity against avian influenza viruses in chickens

Chitosan nanoparticles (CNPs) represent an efficient vaccination tool to deliver immunogenic antigens to the antigen-presenting cells (APCs), which subsequently stimulate protective immune responses against infectious diseases. Herein, we prepared CNPs encapsulating mRNA molecules followed by surface coating with conserved H9N2 HA2 and M2e influenza proteins. We demonstrated that CNPs efficiently delivered mRNA molecules into APCs and had effectively penetrated the mucosal barrier to reach to the immune initiation sites. To investigate the potential of CNPs delivering influenza antigens to stimulate protective immunity, we intranasally vaccinated chickens with empty CNPs, CNPs delivering HA2 and M2e in both mRNA and protein formats (CNPs + RNA + Pr) or CNPs delivering antigens in protein format only (CNPs + Pr). Our results demonstrated that chickens vaccinated with CNPs + RNA + Pr elicited significantly (p < 0.05) higher systemic IgG, mucosal IgA antibody responses and cellular immune responses compared to the CNPs + Pr vaccinated group. Consequently, upon challenge with either H7N9 or H9N2 avian influenza viruses (AIVs), efficient protection, in the context of viral load and lung pathology, was observed in chickens vaccinated with CNPs + RNA + Pr than CNPs + Pr vaccinated group. In conclusion, we show that HA2 and M2e antigens elicited a broad spectrum of protection against AIVs and incorporation of mRNAs in vaccine formulation is an effective strategy to induce superior immune responses.

Safety and immunogenicity of the oral, inactivated, enterotoxigenic Escherichia coli vaccine ETVAX in Bangladeshi children and infants: a double-blind, randomised, placebo-controlled phase 1/2 trial

SummaryBackgroundEnterotoxigenic Escherichia coli causes diarrhoea, leading to substantial mortality and morbidity in children, but no specific vaccine exists. This trial tested an oral, inactivated, enterotoxigenic E coli vaccine (ETVAX), which has been previously shown to be safe and highly immuongenic in Swedish and Bangladeshi adults. We tested the safety and immunogenicity of ETVAX, consisting of four E coli strains overexpressing the most prevalent colonisation factors (CFA/I, CS3, CS5, and CS6) and a toxoid (LCTBA) administered with or without a double-mutant heat-labile enterotoxin (dmLT) as an adjuvant, in Bangladeshi children.MethodsWe did a randomised, double-blind, placebo-controlled, dose-escalation, age-descending, phase 1/2 trial in Dhaka, Bangladesh. Healthy children in one of three age groups (24–59 months, 12–23 months, and 6–11 months) were eligible. Children were randomly assigned with block randomisation to receive either ETVAX, with or without dmLT, or placebo. ETVAX (half [5·5 × 1010 cells], quarter [2·5 × 1010 cells], or eighth [1·25 × 1010 cells] adult dose), with or without dmLT adjuvant (2·5 μg, 5·0 μg, or 10·0 μg), or placebo were administered orally in two doses 2 weeks apart. Investigators and participants were masked to treatment allocation. The primary endpoint was safety and tolerability, assessed in all children who received at least one dose of vaccine. Antibody responses to vaccine antigens, defined as at least a two-times increase in antibody levels between baseline and post-immunisation, were assessed as secondary endpoints. This trial is registered with ClinicalTrials.gov, NCT02531802.FindingsBetween Dec 7, 2015, and Jan 10, 2017, we screened 1500 children across the three age groups, of whom 430 were enrolled and randomly assigned to the different treatment groups (130 aged 24–59 months, 100 aged 12–23 months, and 200 aged 6–11 months). All participants received at least one dose of vaccine. No solicited adverse events occurred that were greater than moderate in severity, and most were mild. The most common solicited event was vomiting (ten [8%] of 130 patients aged 24–59 months, 13 [13%] of 100 aged 12–23 months, and 29 [15%] of 200 aged 6–11 months; mostly of mild severity), which appeared related to dose and age. The addition of dmLT did not modify the safety profile. Three serious adverse events occurred but they were not considered related to the study drug. Mucosal IgA antibody responses in lymphocyte secretions were detected against all primary vaccine antigens (CFA/I, CS3, CS5, CS6, and the LCTBA toxoid) in most participants in the two older age groups, whereas such responses to four of the five antigens were less frequent and of lower magnitude in infants aged 6–11 months than in older children. Faecal secretory IgA immune responses were recorded against all vaccine antigens in infants aged 6–11 months. 78 (56%) of 139 infants aged 6–11 months who were vaccinated developed mucosal responses against at least three of the vaccine antigens versus 14 (29%) of 49 of the infants given placebo. Addition of the adjuvant dmLT enhanced the magnitude, breadth, and kinetics (based on number of responders after the first dose of vaccine) of immune responses in infants.InterpretationThe encouraging safety and immunogenicity of ETVAX and benefit of dmLT adjuvant in young children support its further assessment for protective efficacy in children in enterotoxigenic E coli-endemic areas.FundingPATH (Bill & Melinda Gates Foundation and the UK's Department for International Development), the Swedish Research Council, and The Swedish Foundation for Strategic Research.

CpG ODN G9.1 as a novel nasal ODN adjuvant elicits complete protection from influenza virus infection without causing inflammatory immune responses

This study examined the protective efficacy of and immune response to a nasal influenza vaccine combined with a novel mucosal oligodeoxynucleotide (ODN) adjuvant, CpG ODN G9.1 (G9.1), in a model of infection limited to the upper respiratory tract (URT) and a model of infection in the lower respiratory tract (LRT). Mice were nasally primed with an A/California/7/2009 (Cal7) split vaccine (X179A) plus G9.1 and were then nasally given a booster with X179A alone. When mice were challenged with either a large (infection of the LRT) or small (infection limited to the URT) volume of live Cal7 influenza virus, mice nasally given G9.1 combined with X179A had a markedly higher rate of protection against infection limited to the URT. Moreover, this group of mice promptly recovered from an infection of the LRT. When mice were subcutaneously (s.c.) given X179A as a current form of vaccination, they had no protection from an infection limited to the URT but they did recover from an infection of the LRT. The patterns of protection were closely correlated with influenza virus-specific mucosal secretory IgA (SIgA) or serum IgG antibody (Ab) responses. Thus, SIgA Abs responses play an important role in protection from an infection limited to the URT while influenza virus-specific serum IgG Ab responses help to protect from an infection of the LRT. A finding of note is that lungs from mice nasally given G9.1 had low levels of type I IFN-associated protein- and transcription factor-specific mRNA expression. These results suggest that nasal G9.1 can be used as an effective and safe mucosal adjuvant for influenza vaccines since this nasal vaccine system elicits both mucosal SIgA and serum IgG Ab responses that provide complete protection without inducing potent inflammatory responses.

Route of Vaccine Administration Influences the Impact of Fms-Like Tyrosine Kinase 3 Ligand (Flt3L) on Chlamydial-Specific Protective Immune Responses.

We tested the hypothesis that the impact of the Fms-like tyrosine kinase 3-ligand (Flt3L; FL) on recombinant Vibrio cholerae ghost (rVCG) vaccine-induced chlamydial immunity is influenced by route of vaccine delivery. Female C57BL/6J mice were immunized rectally (IR) or intramuscularly (IM) with rVCG co-expressing the Chlamydia trachomatis PmpD and PorB proteins (rVCG- PmpD/PorB) with and without FL or glycoprotein D of HSV-2 (rVCG-gD2) as antigen control. Vaccine evaluation was based on measurement of T cell proliferation, Th1/Th2 cytokine, and humoral responses at systemic and mucosal compartments, and protection against intravaginal challenge infection. Results revealed that high levels of CD4+ T cell-mediated and humoral immune responses, were elicited in mice as a function of both IR and IM immunization. Unexpectedly, co-administration of vaccine with FL enhanced specific Th1-type cytokine levels and T cell proliferative responses following IR but not IM immunization. While administration of vaccine with FL enhanced the specific mucosal and systemic IgA antibody responses following both immunization routes, IgG2c responses were not enhanced following IR delivery. The vaccine-induced immune effectors protected mice against live heterologous C. muridarum infection irrespective of route of vaccine administration, with the regimen incorporating FL having a protective advantage. Further evaluation showed that protection afforded by the FL adjuvanted vaccine was facilitated by CD4+ T cells, as indicated by reduction in the intensity and duration of genital chlamydial shedding by naïve mice following adoptive transfer of immune CD4+ T cells. Taken together, the results indicate that comparable protective immunity, which is enhanced by co-delivery with FL, is elicited in the female genital tract against Chlamydia infection after mucosal and systemic administration, highlighting the ability of FL to function as an effective immunostimulator at both mucosal and systemic sites. The differential modulation of humoral and cellular immune responses, and protective immunity afforded by the FL adjuvanted vaccine following IR administration indicates that the immunomodulatory impact of FL on chlamydial-specific immunity is influenced by the route of vaccine administration. Thus, targeting of VCG-based vaccines to antigen presenting cells by co-delivery with FL is a feasible immunization approach for inducing effective chlamydial immunity in the female genital tract.

Serotype-Independent Protection Against Invasive Pneumococcal Infections Conferred by Live Vaccine With lgt Deletion.

Streptococcus pneumoniae is the most common respiratory bacterial pathogen among cases of community-acquired infection in young children, older adults, and individuals with underlying medical conditions. Although capsular polysaccharide-based pneumococcal vaccines have contributed to significant decrease in invasive pneumococcal infections, these vaccines have some limitations, including limited serotype coverage, lack of effective mucosal antibody responses, and high costs. In this study, we investigated the safety and immunogenicity of a live, whole-cell pneumococcal vaccine constructed by deleting the gene for prolipoprotein diacylglyceryl transferase (lgt) from the encapsulated pneumococcal strain TIGR4 (TIGR4Δlgt) for protection against heterologous pneumococcal strains. Pneumococcal strain TIGR4 was successfully attenuated by deletion of lgt, resulting in the loss of inflammatory activity and virulence. TIGR4Δlgt colonized the nasopharynx long enough to induce strong mucosal IgA and IgG2b-dominant systemic antibody responses that were cross-reactive to heterologous pneumococcal serotypes. Finally, intranasal immunization with TIGR4Δlgt provided serotype-independent protection against pneumococcal challenge in mice. Taken together, our results suggest that TIGR4Δlgt is an avirulent and attractive broad-spectrum pneumococcal vaccine candidate. More broadly, we assert that modulation of such “master” metabolic genes represents an emerging strategy for developing more effective vaccines against numerous infectious agents.

CCR7-deficient mice exhibit a delayed antigen-specific mucosal IgA antibody response to an oral recombinant Salmonella strain.

The migration of antigen (Ag)-loading dendritic cells (DCs) from Peyer's patches (PPs) to the draining mesenteric lymph nodes (MLNs) via chemokine receptor 7 (CCR7) is thought to be an important step in the initiation of acquired immunity. Our previous study showed that PPs were indispensable for Ag-specific secretory (S)IgA antibody (Ab) responses against oral recombinant Salmonella (rSalmonella). In this study, we attempted to show direct PP DC migration to MLNs by employing photoconvertible protein transgenic mice and investigated the role of the CCR7 signaling pathway in mucosal IgA induction. Our results demonstrated an actual flux of DCs from PPs to MLNs. The frequency of CCR7+ CD11c+ DCs in MLNs of PP-deficient mice was reduced, suggesting that some PP DCs migrated via CCR7. Immunization of CCR7-/- mice elicited significantly lower levels of Ag-specific SIgA Ab responses, which was associated with diminished formation of the germinal center in PPs. However, increased SIgA Ab production and dissemination of rSalmonella were observed at later time points. These results suggest that, although CCR7 was required for SIgA induction at normal velocity, the CCR7-mediated pathway is not essential for the induction of Ag-specific SIgA Ab responses to rSalmonella.

Mucosal Immunity and Protective Efficacy of Intranasal Inactivated Influenza Vaccine Is Improved by Chitosan Nanoparticle Delivery in Pigs.

Annually, swine influenza A virus (SwIAV) causes severe economic loss to swine industry. Currently used inactivated SwIAV vaccines administered by intramuscular injection provide homologous protection, but limited heterologous protection against constantly evolving field viruses, attributable to the induction of inadequate levels of mucosal IgA and cellular immune responses in the respiratory tract. A novel vaccine delivery platform using mucoadhesive chitosan nanoparticles (CNPs) administered through intranasal (IN) route has the potential to elicit strong mucosal and systemic immune responses in pigs. In this study, we evaluated the immune responses and cross-protective efficacy of IN chitosan encapsulated inactivated SwIAV vaccine in pigs. Killed SwIAV H1N2 (δ-lineage) antigens (KAg) were encapsulated in chitosan polymer-based nanoparticles (CNPs-KAg). The candidate vaccine was administered twice IN as mist to nursery pigs. Vaccinates and controls were then challenged with a zoonotic and virulent heterologous SwIAV H1N1 (γ-lineage). Pigs vaccinated with CNPs-KAg exhibited an enhanced IgG serum antibody and mucosal secretory IgA antibody responses in nasal swabs, bronchoalveolar lavage (BAL) fluids, and lung lysates that were reactive against homologous (H1N2), heterologous (H1N1), and heterosubtypic (H3N2) influenza A virus strains. Prior to challenge, an increased frequency of cytotoxic T lymphocytes, antigen-specific lymphocyte proliferation, and recall IFN-γ secretion by restimulated peripheral blood mononuclear cells in CNPs-KAg compared to control KAg vaccinates were observed. In CNPs-KAg vaccinated pigs challenged with heterologous virus reduced severity of macroscopic and microscopic influenza-associated pulmonary lesions were observed. Importantly, the infectious SwIAV titers in nasal swabs [days post-challenge (DPC) 4] and BAL fluid (DPC 6) were significantly (p < 0.05) reduced in CNPs-KAg vaccinates but not in KAg vaccinates when compared to the unvaccinated challenge controls. As well, an increased frequency of T helper memory cells and increased levels of recall IFNγ secretion by tracheobronchial lymph nodes cells were observed. In summary, chitosan SwIAV nanovaccine delivered by IN route elicited strong cross-reactive mucosal IgA and cellular immune responses in the respiratory tract that resulted in a reduced nasal viral shedding and lung virus titers in pigs. Thus, chitosan-based influenza nanovaccine may be an ideal candidate vaccine for use in pigs, and pig is a useful animal model for preclinical testing of particulate IN human influenza vaccines.

Efficacy and synergy of live-attenuated and inactivated influenza vaccines in young chickens.

Outbreaks of novel highly pathogenic avian influenza viruses have been reported in poultry species in the United States since 2014. These outbreaks have proven the limitations of biosecurity control programs, and new tools are needed to reinforce the current avian influenza control arsenal. Some enzootic countries have implemented inactivated influenza vaccine (IIV) in their control programs, but there are serious concerns that a long-term use of IIV without eradication may result in the selection of novel antigenically divergent strains. A broadly protective vaccine is needed, such as live-attenuated influenza vaccine (LAIV). We showed in our previous studies that pc4-LAIV (a variant that encodes a C-terminally truncated NS1 protein) can provide significant protection against heterologous challenge virus in chickens vaccinated at 2–4 weeks of age through upregulation of innate and adaptive immune responses. The current study was conducted to compare the performances of pc4-LAIV and IIV in young chickens vaccinated at 1 day of age. A single dose of pc4-LAIV was able to induce stronger innate and mucosal IgA responses and protect young immunologically immature chickens better than a single dose of IIV. Most importantly, when 1-day-old chickens were intranasally primed with pc4-LAIV and subcutaneously boosted with IIV three weeks later, they showed a rapid, robust, and highly cross-reactive serum antibody response and a high level of mucosal IgA antibody response. This vaccination regimen warrants further optimization to increase its range of protection.