Oral Biologic Delivery: Advances Toward Oral Subunit, DNA, and mRNA Vaccines and the Potential for Mass Vaccination During Pandemics.

Oral vaccination enables pain-free and self-administrable vaccine delivery for rapid mass vaccination during pandemic outbreaks. Furthermore, it elicits systemic and mucosal immune responses. This protects against infection at mucosal surfaces, which may further enhance protection and minimize the spread of disease. The gastrointestinal (GI) tract presents a number of prospective mucosal inductive sites for vaccine targeting, including the oral cavity, stomach, and small intestine. However, currently available oral vaccines are effectively limited to live-attenuated and inactivated vaccines against enteric diseases. The GI tract poses a number of challenges,including degradative processes that digest biologics and mucosal barriers that limit their absorption. This review summarizes the approaches currently under development and future opportunities for oral vaccine delivery to established (intestinal) and relatively new (oral cavity, stomach) mucosal targets. Special consideration is given to recent advances in oral biologic delivery that offer promise as future platforms for the administration of oral vaccines.

Induction of Transmucosal Protection by Oral Vaccination with an Attenuated Chlamydia.

Chlamydia muridarum has been used to study chlamydial pathogenesis because it induces mice to develop hydrosalpinx, a pathology observed in C. trachomatis-infected women. We identified a C. muridarum mutant that is no longer able to induce hydrosalpinx. In the current study, we evaluated the mutant as an attenuated vaccine. Following an intravaginal immunization with the mutant, mice were protected from hydrosalpinx induced by wild-type C. muridarum. However, the mutant itself productively colonized the mouse genital tract and produced infectious organisms in vaginal swabs. Nevertheless, the mutant failed to produce infectious shedding in the rectal swabs following an oral inoculation. Importantly, mice orally inoculated with the mutant mounted transmucosal immunity against challenge infection of wild-type C. muridarum in the genital tract. The protection was detected as early as day 3 following the genital challenge infection and the orally immunized mice were protected from any significant pathology in the upper genital tract. However, the same orally immunized mice failed to prevent the colonization of wild-type C. muridarum in the gastrointestinal tract. The transmucosal immunity induced by the oral mutant was further validated in the airway. The orally vaccinated mice were protected from both lung infection and systemic toxicity caused by intranasally inoculated wild-type C. muridarum although the same mice still permitted the gastrointestinal colonization by the wild-type C. muridarum. These observations suggest that the mutant C. muridarum may be developed into an intracellular oral vaccine vector (or IntrOv) for selectively inducing transmucosal immunity in extra-gut tissues.

Transnuclear mice reveal Peyer's patch iNKT cells that regulate B-cell class switching to IgG1.

Tissue-resident iNKT cells maintain tissue homeostasis and peripheral surveillance against pathogens; however, studying these cells is challenging due to their low abundance and poor recovery from tissues. We here show that iNKT transnuclear mice, generated by somatic cell nuclear transfer, have increased tissue resident iNKT cells. We examined expression of PLZF, T-bet, and RORγt, as well as cytokine/chemokine profiles, and found that both monoclonal and polyclonal iNKT cells differentiated into functional subsets that faithfully replicated those seen in wild-type mice. We detected iNKT cells from tissues in which they are rare, including adipose, lung, skin-draining lymph nodes, and a previously undescribed population in Peyer's patches (PP). PP-NKT cells produce the majority of the IL-4 in Peyer's patches and provide indirect help for B-cell class switching to IgG1 in both transnuclear and wild-type mice. Oral vaccination with α-galactosylceramide shows enhanced fecal IgG1 titers in iNKT cell-sufficient mice. Transcriptional profiling reveals a unique signature of PP-NKT cells, characterized by tissue residency. We thus define PP-NKT as potentially important for surveillance for mucosal pathogens.

Generation of Recombinant Rotaviruses Expressing Human Norovirus Capsid Proteins.

Rotavirus, a segmented double-stranded RNA virus of the Reoviridae family, is a primary cause of acute gastroenteritis in young children. In countries where rotavirus vaccines are widely used, norovirus (NoV) has emerged as the major cause of acute gastroenteritis. Towards the goal of creating a combined rotavirus-NoV vaccine, we explored the possibility of generating recombinant rotaviruses (rRVs) expressing all or portions of the NoV GII.4 VP1 capsid protein. This was accomplished by replacing the segment 7 NSP3 open reading frame with a cassette encoding, sequentially, NSP3, a 2A stop-restart translation element, and all or portions (P, P2) of NoV VP1. In addition to successfully recovering rRVs with modified SA11 segment 7 RNAs encoding NoV capsid proteins, analogous rRVs were recovered through modification of the segment 7 RNA of the RIX4414 vaccine strain. An immunoblot assay confirmed that rRVs expressed NoV capsid proteins as independent products. Moreover, VP1 expressed by rRVs underwent dimerization and was recognized by conformational-dependent anti-VP1 antibodies. Serially passaged rRVs that expressed the NoV P and P2 were genetically stable, retaining additional sequences of up to 1.1 kbp without change. However, serially passaged rRVs containing the longer 1.6-kb VP1 sequence were less stable and gave rise to virus populations with segment 7 RNAs lacking VP1 coding sequences. Together, these studies suggest that it may be possible to develop combined rotavirus-NoV vaccines using modified segment 7 RNA to express NoV P or P2. In contrast, development of potential rotavirus-NoV vaccines expressing NoV VP1 will need additional efforts to improve genetic stability. IMPORTANCE Rotavirus (RV) and norovirus (NoV) are the two most important causes of acute viral gastroenteritis (AGE) in infants and young children. While the incidence of RV AGE has been brought under control in many countries through the introduction of universal mass vaccination with live attenuated RV vaccines, similar highly effective NoV vaccines are not available. To pursue the development of a combined RV-NoV vaccine, we examined the potential of using RV as an expression vector of all or portions of the NoV capsid protein VP1. Our results showed that by replacing the NSP3 open reading frame in RV genome segment 7 RNA with a coding cassette for NSP3, a 2A stop-restart translation element, and VP1, recombinant RVs can be generated that express NoV capsid proteins. These findings raise the possibility of developing new generations of RV-based combination vaccines that provide protection against a second enteric pathogen, such as NoV.

An updated review on oral protein-based antigen vaccines efficiency and delivery approaches: a special attention to infectious diseases.

Various infectious agents affect human health via the oral entrance. The majority of pathogens lack approved vaccines. Oral vaccination is a convenient, safe and cost-effective approach with the potential of provoking mucosal and systemic immunity and maintaining individual satisfaction. However, vaccines should overcome the intricate environment of the gastrointestinal tract (GIT). Oral protein-based antigen vaccines (OPAVs) are easier to administer than injectable vaccines and do not require trained healthcare professionals. Additionally, the risk of needle-related injuries, pain, and discomfort is eliminated. However, OPAVs stability at environmental and GIT conditions should be considered to enhance their stability and facilitate their transport and storage. These vaccines elicit the local immunity, protecting GIT, genital tract and respiratory epithelial surfaces, where numerous pathogens penetrate the body. OPAVs can also be manipulated (such as using specific incorporated ligand and receptors) to elicit targeted immune response. However, low bioavailability of OPAVs necessitates development of proper protein carriers and formulations to enhance their stability and efficacy. There are several strategies to improve their efficacy or protective effects, such as incorporation of adjuvants, enzyme inhibitors, mucoadhesive or penetrating devices and permeation enhancers. Hence, efficient delivery of OPAVs into GIT require proper delivery systems mainly including smart target systems, probiotics, muco-adhesive carriers, lipid- and plant-based delivery systems and nano- and microparticles.

An improved oral vaccine with molecular adjuvant ß-defensin protects grouper against nervous necrosis virus infection.

Nervous necrosis virus (NNV) is one of the most important fish viral pathogens infecting more than 120 fish species worldwide. Due to the mass mortality rates often seen among larvae and juveniles, few effective vaccines against NNV were developed up to now. Here, the protective effect of recombinant coat protein (CP) from red-spotted grouper nervous necrosis virus (RGNNV) fused with grouper ß-defensin (DEFB) as an oral vaccine was evaluated using Artemia as a biocarrier delivery system in pearl gentian grouper (Epinephelus lanceolatus♂ × Epinephelus fuscoguttatus♀). Feeding with Artemia encapsulated with E. coli expressing control vector (control group), CP, or CP-DEFB showed no obvious side effects on the growth of groupers. ELISA and antibody neutralization assay showed that CP-DEFB oral vaccination group induced higher anti-RGNNV CP specific antibodies and exhibited higher neutralization potency than the CP and control group. Meanwhile, the expression levels of several immune and inflammatory factors in the spleen and kidney after feeding with CP-DEFB were also significantly increased compared with the CP group. Consistently, after challenge with RGNNV, groupers fed CP-DEFB and CP exhibited 100% and 88.23% relative percentage survival (RPS), respectively. Moreover, the lower transcription levels of viral genes and milder pathological changes in CP-DEFB group were detected compared with the CP and control group. Thus, we proposed that grouper ß-defensin functioned as an efficient molecular adjuvant for an improved oral vaccine against nervous necrosis virus infection.

Mucosal and systemic immune effects of Bacillus subtilis in rainbow trout (Oncorhynchus mykiss).

Bacillus spp. are well known for their probiotic properties. Hence, the long-term feeding of Bacillus spp. strains to different fish species has been proved to confer beneficial effects regarding growth or pathogen resistance, among others. However, whether these strains could function as mucosal adjuvants, up-regulating immune responses after a single administration, has not yet been investigated in fish. Thus, in the current work, we have performed a series of experiments in rainbow trout (Oncorhynchus mykiss) aimed at establishing the potential of two Bacillus subtilis spore-forming strains, designated as ABP1 and ABP2, as oral adjuvants/immunostimulants. As an initial step, we evaluated their transcriptional effects on the rainbow trout intestinal epithelial cell line RTgutGC, and in gut tissue explants incubated ex vivo with the two strains. Their capacity to adhere to RTgutGC cells was also evaluated by flow cytometry. Although both strains had the capacity to modulate the transcription of several genes related to innate and adaptive immune responses, it was the ABP1 strain that led to stronger transcriptional effects, also exerting a higher binding capacity to intestinal epithelial cells. Consequently, we selected this strain to establish its effects on splenic B cells upon in vitro exposure as well as to determine the transcriptional effects exerted in the spleen, kidney, and gut after a single oral administration of the bacteria. Our results showed that B. subtilis ABP1 had the capacity to modulate the proliferation, IgM secreting capacity and MHC II surface expression of splenic B cells. Finally, we confirmed that this strain also induced the transcription of genes involved in inflammation, antimicrobial genes, and genes involved in T cell responses upon a single oral administration. Our results provide valuable information regarding how B. subtilis modulates the immune response of rainbow trout, pointing to the usefulness of the ABP1 strain to design novel oral vaccination strategies for aquaculture.

A guide to oral vaccination: Highlighting electrospraying as a promising manufacturing technique toward a successful oral vaccine development.

Most vaccines approved by regulatory bodies are administered via intramuscular or subcutaneous injections and have shortcomings, such as the risk of needle-associated blood infections, pain and swelling at the injection site. Orally administered vaccines are of interest, as they elicit both systemic and mucosal immunities, in which mucosal immunity would neutralize the mucosa invading pathogen before the onset of an infection. Hence, oral vaccination can eliminate the injection associated adverse effects and enhance the person's compliance. Conventional approaches to manufacturing oral vaccines, such as coacervation, spray drying, and membrane emulsification, tend to alter the structural proteins in vaccines that result from high temperature, organic and toxic solvents during production. Electrohydrodynamic processes, specifically electrospraying, could solve these challenges, as it also modulates antigen release and has a high loading efficiency. This review will highlight the mucosal immunity and biological basis of the gastrointestinal immune system, different oral vaccine delivery approaches, and the application of electrospraying in vaccines development.

Immunogenicity and Efficacy of Live-Attenuated Salmonella Typhimurium Vaccine Candidate CVD 1926 in a Rhesus Macaque Model of Gastroenteritis.

Salmonella Typhimurium is a common cause of foodborne gastroenteritis and a less frequent but important cause of invasive disease, especially in developing countries. In our previous work, we showed that a live-attenuated S. Typhimurium vaccine (CVD 1921) was safe and immunogenic in rhesus macaques, although shed for an unacceptably long period (10 days) postimmunization. Consequently, we engineered a new strain, CVD 1926, which was shown to be safe and immunogenic in mice, as well as less reactogenic in mice and human cell-derived organoids than CVD 1921. In this study, we assessed the reactogenicity and efficacy of CVD 1926 in rhesus macaques. Animals were given two doses of either CVD 1926 or saline perorally. The vaccine was well-tolerated, with shedding in stool limited to a mean of 5 days. All CVD 1926-immunized animals had both a serological and a T cell response to vaccination. At 4 weeks postimmunization, animals were challenged with wild-type S. Typhimurium I77. Unvaccinated (saline) animals had severe diarrhea, with two animals succumbing to infection. Animals receiving CVD 1926 were largely protected, with only one animal having moderate diarrhea. Vaccine efficacy in this gastroenteritis model was 80%. S. Typhimurium vaccine strain CVD 1926 was safe and effective in rhesus macaques and shed for a shorter period than other previously tested live-attenuated vaccine strains. This strain could be combined with other live-attenuated Salmonella vaccine strains to create a pan-Salmonella vaccine.

Serial Passaging of the Human Rotavirus CDC-9 Strain in Cell Culture Leads to Attenuation: Characterization from In Vitro and In Vivo Studies.

Live oral rotavirus vaccines have been developed by serial passaging in cell culture and found to be safe in infants. However, mechanisms for the adaptation and attenuation of rotavirus vaccines are not fully understood. We prepared a human rotavirus vaccine strain, CDC-9 (G1P[8]), which when grown in MA104 cells to passage 11 or 12 (P11/P12) had no nucleotide or amino acid sequence changes from the original virus in stool. Upon adaptation and passages in Vero cells, the strain underwent five amino acid changes at P28 and one additional change at P44/P45 in the VP4 gene. We performed virologic, immunological, and pathogenic characterization of wild-type CDC-9 virus at P11/P12 and its two mutants at P28 or P44/P45 using in vitro and in vivo model systems. We found that mutants CDC-9 P28 and P44 induced upregulated expression of immunomodulatory cytokines. On the other hand, the two mutant viruses induced lower STAT1 phosphorylation and grew to 2-log-higher titers than wild-type virus in human Caco-2 cells and simian Vero cells. In neonatal rats, CDC-9 P45 showed reduced rotavirus shedding in fecal specimens and did not induce diarrhea compared to wild-type virus and modulated cytokine responses comparably to Rotarix infection. These findings indicate that mutant CDC-9 is attenuated and safe. Our study is the first to provide insight into the possible mechanisms of human rotavirus adaptation and attenuation and supports ongoing efforts to develop CDC-9 as a new generation of rotavirus vaccine for live oral or parenteral administration.IMPORTANCE Mechanisms for in vitro adaptation and in vivo attenuation of human rotavirus vaccines are not known. The present study is the first to comprehensively compare the in vitro growth characteristics, virulence, and host response of a wild-type and an attenuated human rotavirus strain, CDC-9, in Caco-2 cells and neonatal rats. Our study identifies critical sequence changes in the genome that render human rotavirus adapted to growth to high levels in Vero cells and attenuated and safe in neonatal rats; thus, the study supports clinical development of CDC-9 for oral or parenteral vaccination in children.

Immune evaluation of a Saccharomyces cerevisiae-based oral vaccine against Helicobacter pylori in mice.

BACKGROUND: Helicobacter pylori (H. pylori) is a common human pathogenic bacterium that is associated with gastric diseases. The current leading clinical therapy is combination antibiotics, but this treatment has safety issues, especially the induction of drug resistance. Therefore, developing a safe and effective vaccine against H. pylori is one of the best alternatives. OBJECTIVE: To develop Saccharomyces cerevisiae (S. cerevisiae)-based oral vaccines and then demonstrate the feasibility of this platform for preventing H. pylori infection in the absence of a mucosal adjuvant. MATERIALS AND METHODS: Saccharomyces cerevisiae (S. cerevisiae)-based oral vaccines, including EBY100/pYD1-UreB and EBY100/pYD1-VacA, were generated and analyzed by Western blot, Immunofluorescence analysis, flow cytometric assay, and indirect enzyme-link immunosorbent assay (ELISA). Further, antibody responses induced by oral administration of EBY100/pYD1-UreB, EBY100/pYD1-VacA, or EBY100/pYD1-UreB + EBY100/pYD1-VacA were measured in a mouse model. Lastly, the vaccinated mice were infected with H. pylori SS1, and colonization in the stomach were evaluated. RESULTS: Saccharomyces cerevisiae-based H. pylori oral vaccines were successfully constructed. Mice orally administered with EBY100/pYD1-UreB, EBY100/pYD1-VacA, or EBY100/pYD1-UreB + EBY100/pYD1-VacA exhibited a significant humoral immune response as well as a mucosal immune response. Importantly, S. cerevisiae-based oral vaccines could effectively reduce bacterial loads with statistical significance after H. pylori infection. CONCLUSIONS: Our study shows that S. cerevisiae-based platforms can serve as an alternative approach for the future development of promising bacterial oral vaccine candidates.

Current status and future prospects of multi-antigen tick vaccine.

Ticks are blood sucking ectoparasite that transmit several pathogens to humans and animals. Tick management focusing on use of chemicals has several drawbacks including development of multi-acaricide resistant tick populations. To minimize the use of chemicals on animals and on the environment, immunization of natural hosts is considered a viable component of Integrated Tick Management System. Most of the tick vaccine trials are focused on single antigen immunization directed against homologous challenge. From commercial point of view, vaccination against one given tick species is not a feasible option. In this context, multi-antigen vaccines comprising of candidate antigens of multiple tick species or both ticks and tick-borne pathogens have commercial potential. Different strategies are considered for the development of multi-antigen tick and/or tick-borne pathogen vaccines. Further, the efficacy of vaccine can be improved by adopting the 'omics' tools and techniques in selection of novel antigens and efficient delivery like Lipid Nano Particle (LNP)-mRNA vaccines, viral vector vaccine, live vector vaccine etc. into the host. The subject has been reviewed to address the current status of multi antigen tick vaccines and formulations of the future strategies for the control of TTBDs of human and animals.

Potential of the Escherichia coli LT(R192G/L211A) toxoid as a mucosal adjuvant for rainbow trout (Oncorhynchus mykiss).

Oral vaccines are highly demanded by aquaculture sector that requires alternatives to injectable vaccines, involving fish handling, stress-related immunosuppression and mortalities. However, most previous attempts to obtain effective oral vaccines have failed due to a restricted tolerance mechanisms in intestine, whose mucosa is at the frontline of antigen encounter and has to balance the equilibrium between tolerance and immunity in a microbe-rich environment. Thus, the search for oral adjuvants that could augment immune responses triggered by antigens allowing them to circumvent intestinal tolerance is of great relevance. The present work focuses on the adjuvant potential of the Escherichia coli LT(R192G/L211A) toxoid (dmLT). To undertake an initial screening of the potential that dmLT has as an oral adjuvant in rainbow trout (Oncorhynchus mykiss), we have analyzed its transcriptional effects alone or in combination with Aeromonas salmonicida subsp. salmonicida or viral hemorrhagic septicemia virus (VHSV) on rainbow trout intestinal epithelial cell line RTgutGC and gut explants. Our results show that although dmLT provoked no significant effects by itself, it increased the transcription of pro-inflammatory cytokines and antimicrobial genes induced by the bacteria. In contrast, when combined with VHSV, dmLT only increased the transcription of Mx and the intracellular adhesion molecule 1 (ICAM1). Therefore, the protocol designed is an effective method to initially evaluate the effects of potential oral adjuvants, and points to dmLT as an effective adjuvant for oral antibacterial vaccines.

Genome-Wide Identification of Virulence Genes in Erysipelothrix rhusiopathiae: Use of a Mutant Deficient in a tagF Homolog as a Safe Oral Vaccine against Swine Erysipelas.

Swine erysipelas is caused by the Gram-positive pathogen Erysipelothrix rhusiopathiae The swine erysipelas live vaccine in Japan, the E. rhusiopathiae Koganei 65-0.15 strain (Koganei), has been reported to cause arthritis and endocarditis. To develop a vaccine with increased safety, we used a virulent Fujisawa strain to construct transposon mutants for a total of 651 genes, which covered 38% of the coding sequence of the genome. We screened the mutants for attenuation by inoculating mice with 108 CFU of each mutant and subsequently assessed protective capability by challenging the surviving mice with 103 CFU (102 times the 50% lethal dose) of the Fujisawa strain. Of the 23 attenuated mutants obtained, 6 mutants were selected and evaluated for protective capability in pigs by comparison to that of the Koganei strain. A mutant in the ERH_0432 (tagF) gene encoding a putative CDP-glycerol glycerophosphotransferase was found to be highly attenuated and to induce humoral and cell-mediated immune responses in conventional pigs. An in-frame deletion mutant of the gene, the Δ432 mutant, was constructed, and attenuation was further confirmed in germfree piglets; three of four piglets subcutaneously inoculated with 109 CFU of the Δ432 mutant showed no apparent clinical symptoms, whereas all four of the Koganei-inoculated piglets died 3 days after inoculation. It was confirmed that conventional pigs inoculated orally or subcutaneously with the Δ432 strain were almost completely protected against lethal challenge infection. Thus, the tagF homolog mutant of E. rhusiopathiae represents a safe vaccine candidate that can be administered via the oral and subcutaneous routes.

Intragastric Administration of Lactobacillus plantarum and 2,2'-Dithiodipyridine-Inactivated Simian Immunodeficiency Virus (SIV) Does Not Protect Indian Rhesus Macaques from Intrarectal SIV Challenge or Reduce Virus Replication after Transmission.

A major obstacle to development of an effective AIDS vaccine is that along with the intended beneficial responses, the immunization regimen may activate CD4+ T cells that can facilitate acquisition of human immunodeficiency virus (HIV) by serving as target cells for the virus. Lu et al. (W. Lu et al., Cell Rep 2:1736-1746, 2012, https://doi.org/10.1016/j.celrep.2012.11.016) reported that intragastric administration of chemically inactivated simian immunodeficiency virus SIVmac239 and Lactobacillus plantarum (iSIV-L. plantarum) protected 15/16 Chinese-origin rhesus macaques (RMs) from high-dose intrarectal SIVmac239 challenge at 3 months postimmunization. They attributed the observed protection to induction of immune tolerance, mediated by "MHC-Ib/E-restricted CD8+ regulatory T cells that suppressed SIV-harboring CD4+ T cell activation and ex vivo SIV replication in 15/16 animals without inducing SIV-specific antibodies or cytotoxic T." J.-M. Andrieu et al. (Front Immunol 5:297, 2014, https://doi.org/10.3389/fimmu.2014.00297) subsequently reported protection from infection in 23/24 RMs immunized intragastrically or intravaginally with iSIV and Mycobacterium bovis BCG, L. plantarum, or Lactobacillus rhamnosus, which they ascribed to the same tolerogenic mechanism. Using vaccine materials obtained from our coauthors, we conducted an immunization and challenge experiment with 54 Indian RMs and included control groups receiving iSIV only or L. plantarum only as well as unvaccinated animals. Intrarectal challenge with SIVmac239 resulted in rapid infection in all groups of vaccinated RMs as well as unvaccinated controls. iSIV-L. plantarum-vaccinated animals that became SIV infected showed viral loads similar to those observed in animals receiving iSIV only or L. plantarum only or in unvaccinated controls. The protection from SIV transmission conferred by intragastric iSIV-L. plantarum administration reported previously for Chinese-origin RMs was not observed when the same experiment was conducted in a larger cohort of Indian-origin animals.IMPORTANCE Despite an increased understanding of immune responses against HIV, a safe and effective AIDS vaccine is not yet available. One obstacle is that immunization may activate CD4+ T cells that may act as target cells for acquisition of HIV. An alternative strategy may involve induction of a tolerance-inducing response that limits the availability of activated CD4+ T cells, thus limiting the ability of virus to establish infection. In this regard, exciting results were obtained for Chinese-origin rhesus macaques by using a "tolerogenic" vaccine, consisting of intragastric administration of Lactobacillus plantarum and 2,2'-dithiodipyridine-inactivated SIV, which showed highly significant protection from virus transmission. In the present study, we administered iSIV-L. plantarum to Indian-origin rhesus macaques and failed to observe any protective effect on virus acquisition in this experimental setting. This work is important because it contributes to the overall assessment of the clinical potential of a new candidate AIDS vaccine platform based on iSIV-L. plantarum.

Recombinant nodavirus vaccine produced in bacteria and administered without purification elicits humoral immunity and protects European sea bass against infection.

Viral necrosis virus (NNV) or nodavirus causes fish viral encephalopathy and retinopathy worldwide. In some cases, mortalities in aquaculture industry can reach up to 100%, some species being especially sensitive as is the case of European sea bass (Dicentrarchus labrax), one of the main cultured species in the Mediterranean, with the consequent economical loses. Development of new vaccines against NNV is in the spotlight though few researches have focused in European sea bass. In this study we have generated a recombinant NNV (rNNV) vaccine produced in Escherichia coli expressing the capsid protein and administered it to European sea bass juveniles by two different routes (intraperitoneal and oral). The last being considered non-stressful and desired for fish farming of small fish, which in fact are the most affected by NNV. Oral vaccine was composed of feed pellets containing the recombinant whole bacteria, and injected vaccine was composed of recombinant bacteria previously lysed. Our results revealed production of specific anti-NNV IgM following the two vaccination procedures, levels that were further increased in orally-vaccinated group after challenge with NNV. Genes related to interferon (IFN), T-cell and immunoglobulin markers were scarcely regulated in head-kidney (HK), gut or brain. Vaccination by either route elicited a relative survival response of 100% after NNV challenge. To our knowledge, this is the first report of a recombinant vaccine followed by no purification steps which resulted in a complete protection in European sea bass when challenged with NNV.

Improved Tolerability of a Salmonella enterica Serovar Typhimurium Live-Attenuated Vaccine Strain Achieved by Balancing Inflammatory Potential with Immunogenicity.

A notable proportion of Salmonella-associated gastroenteritis in the United States is attributed to Salmonella enterica serovar Typhimurium. We have previously shown that live-attenuated S Typhimurium vaccine candidate CVD 1921 (I77 ΔguaBA ΔclpP) was safe and immunogenic in rhesus macaques but was shed for an undesirably long time postimmunization. In mice, occasional mortality postvaccination was also noted (approximately 1 in every 15 mice). Here we describe a further attenuated vaccine candidate strain harboring deletions in two additional genes, htrA and pipA We determined that S Typhimurium requires pipA to elicit fluid accumulation in a rabbit ileal loop model of gastroenteritis, as an S Typhimurium ΔpipA mutant induced significantly less fluid accumulation in rabbit loops than the wild-type strain. New vaccine strain CVD 1926 (I77 ΔguaBA ΔclpP ΔpipA ΔhtrA) was assessed for inflammatory potential in an organoid model of human intestinal mucosa, where it induced less inflammatory cytokine production than organoids exposed to the precursor vaccine, CVD 1921. To assess vaccine safety and efficacy, mice were given three doses of CVD 1926 (109 CFU/dose) by oral gavage, and at 1 or 3 months postimmunization, mice were challenged with 700 or 100 LD50 (50% lethal doses), respectively, of wild-type strain I77. CVD 1926 was well tolerated and exhibited 47% vaccine efficacy following challenge with a high inoculum and 60% efficacy after challenge with a low inoculum of virulent S Typhimurium. CVD 1926 is less reactogenic yet equally as immunogenic and protective as previous iterations in a mouse model.

Nonpathogenic Colonization with Chlamydia in the Gastrointestinal Tract as Oral Vaccination for Inducing Transmucosal Protection.

Chlamydia has been detected in the gastrointestinal tracts of humans and animals. We now report that gastrointestinal Chlamydia muridarum is able to induce robust transmucosal protection in mice. C. muridarum colonization in the gastrointestinal tract correlated with both a shortened course of C. muridarum genital tract infection and stronger protection against subsequent genital tract challenge infection. Mice preinoculated intragastrically with C. muridarum became highly resistant to subsequent C. muridarum infection in the genital tract, resulting in prevention of pathology in the upper genital tract. The transmucosal protection in the genital tract was rapidly induced, durable, and dependent on major histocompatibility complex (MHC) class II antigen presentation but not MHC class I antigen presentation. Although a deficiency in CD4+ T cells only partially reduced the transmucosal protection, depletion of CD4+ T cells from B cell-deficient mice completely abolished the protection, suggesting a synergistic role of both CD4+ T and B cells in the gastrointestinal C. muridarum-induced transmucosal immunity. However, the same protective immunity did not significantly affect C. muridarum colonization in the gastrointestinal tract. The long-lasting colonization with C. muridarum was restricted to the gastrointestinal tract and was nonpathogenic to either gastrointestinal or extragastrointestinal tissues. Furthermore, gastrointestinal C. muridarum did not alter the gut microbiota or the development of gut mucosal resident memory T cell responses to a nonchlamydial infection. Thus, Chlamydia may be developed into a safe and orally deliverable replicating vaccine for inducing transmucosal protection.

Mucosal Antibodies to the C Terminus of Toxin A Prevent Colonization of Clostridium difficile.

Mucosal immunity is considered important for protection against Clostridium difficile infection (CDI). We show that in hamsters immunized with Bacillus subtilis spores expressing a carboxy-terminal segment (TcdA26-39) of C. difficile toxin A, no colonization occurs in protected animals when challenged with C. difficile strain 630. In contrast, animals immunized with toxoids showed no protection and remained fully colonized. Along with neutralizing toxins, antibodies to TcdA26-39 (but not to toxoids), whether raised to the recombinant protein or to TcdA26-39 expressed on the B. subtilis spore surface, cross-react with a number of seemingly unrelated proteins expressed on the vegetative cell surface or spore coat of C. difficile These include two dehydrogenases, AdhE1 and LdhA, as well as the CdeC protein that is present on the spore. Anti-TcdA26-39 mucosal antibodies obtained following immunization with recombinant B. subtilis spores were able to reduce the adhesion of C. difficile to mucus-producing intestinal cells. This cross-reaction is intriguing yet important since it illustrates the importance of mucosal immunity for complete protection against CDI.

Detection of Emerging Vaccine-Related Polioviruses by Deep Sequencing.

Oral poliovirus vaccine can mutate to regain neurovirulence. To date, evaluation of these mutations has been performed primarily on culture-enriched isolates by using conventional Sanger sequencing. We therefore developed a culture-independent, deep-sequencing method targeting the 5' untranslated region (UTR) and P1 genomic region to characterize vaccine-related poliovirus variants. Error analysis of the deep-sequencing method demonstrated reliable detection of poliovirus mutations at levels of <1%, depending on read depth. Sequencing of viral nucleic acids from the stool of vaccinated, asymptomatic children and their close contacts collected during a prospective cohort study in Veracruz, Mexico, revealed no vaccine-derived polioviruses. This was expected given that the longest duration between sequenced sample collection and the end of the most recent national immunization week was 66 days. However, we identified many low-level variants (<5%) distributed across the 5' UTR and P1 genomic region in all three Sabin serotypes, as well as vaccine-related viruses with multiple canonical mutations associated with phenotypic reversion present at high levels (>90%). These results suggest that monitoring emerging vaccine-related poliovirus variants by deep sequencing may aid in the poliovirus endgame and efforts to ensure global polio eradication.