COVID-19: to vaccinate or not to vaccinate - that is the question.

SARS-CoV-2 is a virus which infects the respiratory tract and may cause severe, occasionally life-threatening disease COVID-19. In more than 5% of symptomatic patients the infection is associated with post-acute symptoms. The initial contact of the virus with the immune system of the nasopharynx and oropharynx induces a mucosal immune response manifested by the production of secretory IgA (sIgA) antibodies which may contribute to the restriction of the infection to the upper respiratory tract and an asymptomatic or clinically mild disease. The current systemically administered vaccines protected against the severe COVID-19 infection and its post-acute sequelae. However, they do not induce antibodies in mucosal secretions in SARS-CoV-2-naive individuals. In contrast, in those who previously experienced mucosal infection, systemically administered vaccines may stimulate sIgA production. The clinical benefit of systemic vaccination convincingly documented in tens of millions of individuals overshadows the rare, sometimes controversial reports of complications encountered after vaccination. The inability of current SARS-CoV-2 vaccines to induce mucosal immune responses and to prevent the spreading of the virus by external secretions demonstrates the mutual independence of mucosal and systemic compartments of the immune system, and thus emphasizes need for the development of vaccines inducing protective immune responses in both compartments.

Diversification of a Novel α-Galactosyl Ceramide Hotspot Boosts the Adjuvant Properties in Parenteral and Mucosal Vaccines.

The development of potent adjuvants is an important step for improving the performance of subunit vaccines. CD1d agonists, such as the prototypical α-galactosyl ceramide (α-GalCer), are of special interest due to their ability to activate iNKT cells and trigger rapid dendritic cell maturation and B-cell activation. Herein, we introduce a novel derivatization hotspot at the α-GalCer skeleton, namely the N-substituent at the amide bond. The multicomponent diversification of this previously unexplored glycolipid chemotype space permitted the introduction of a variety of extra functionalities that can either potentiate the adjuvant properties or serve as handles for further conjugation to antigens toward the development of self-adjuvanting vaccines. This strategy led to the discovery of compounds eliciting enhanced antigen-specific T cell stimulation and a higher antibody response when delivered by either the parenteral or the mucosal route, as compared to a known potent CD1d agonist. Notably, various functionalized α-GalCer analogues showed a more potent adjuvant effect after intranasal immunization than a PEGylated α-GalCer analogue previously optimized for this purpose. Ultimately, this work could open multiple avenues of opportunity for the use of mucosal vaccines against microbial infections.

Efficacy of a Pseudomonas aeruginosa serogroup O9 vaccine.

There are currently no approved vaccines against the opportunistic pathogen Pseudomonas aeruginosa. Among vaccine targets, the lipopolysaccharide (LPS) O antigen of P. aeruginosa is the most immunodominant protective candidate. There are 20 different O antigens composed of different repeat sugar structures conferring serogroup specificity, and 10 are found most frequently in infection. Thus, one approach to combat infection by P. aeruginosa could be to generate immunity with a vaccine cocktail that includes all these serogroups. Serogroup O9 is 1 of the 10 serogroups commonly found in infection, but it has never been developed into a vaccine, due in part to the acid-labile nature of the O9 polysaccharide. Our laboratory has previously shown that intranasal administration of an attenuated Salmonella strain expressing the P. aeruginosa serogroup O11 LPS O antigen was effective in clearing bacteria and preventing mortality in mice following intranasal challenge with serogroup O11 P. aeruginosa. Consequently, we set out to develop a P. aeruginosa serogroup O9 vaccine using a similar approach. Here, we show that Salmonella expressing serogroup O9 triggered an antibody-mediated immune response following intranasal administration to mice and that it conferred protection from P. aeruginosa serogroup O9 in a murine model of acute pneumonia.

ISCOMs/MPLA-Adjuvanted SDAD Protein Nanoparticles Induce Improved Mucosal Immune Responses and Cross-Protection in Mice.

The epidemics caused by the influenza virus are a serious threat to public health and the economy. Adding appropriate adjuvants to improve immunogenicity and finding effective mucosal vaccines to combat respiratory infection at the portal of virus entry are important strategies to boost protection. In this study, a novel type of core/shell protein nanoparticle consisting of influenza nucleoprotein (NP) as the core and NA1-M2e or NA2-M2e fusion proteins as the coating antigens by SDAD hetero-bifunctional crosslinking is exploited. Immune-stimulating complexes (ISCOMs)/monophosphoryl lipid A (MPLA) adjuvants further boost the NP/NA-M2e SDAD protein nanoparticle-induced immune responses when administered intramuscularly. The ISCOMs/MPLA-adjuvanted protein nanoparticles are delivered through the intranasal route to validate the application as mucosal vaccines. ISCOMs/MPLA-adjuvanted nanoparticles induce significantly strengthened antigen-specific antibody responses, cytokine-secreting splenocytes in the systemic compartment, and higher levels of antigen-specific IgA and IgG in the local mucosa. Meanwhile, significantly expanded lung resident memory (RM) T and B cells (TRM /BRM ) and alveolar macrophages population are observed in ISCOMs/MPLA-adjuvanted nanoparticle-immunized mice with a 100% survival rate after homogeneous and heterogeneous H3N2 viral challenges. Taken together, ISCOMs/MPLA-adjuvanted protein nanoparticles could improve strong systemic and mucosal immune responses conferring protection in different immunization routes.

The unfulfilled potential of mucosal immunization.

Recent events involving the global coronavirus pandemic have focused attention on vaccination strategies. Although tremendous advances have been made in subcutaneous and intramuscular vaccines during this time, one area that has lagged in implementation is mucosal immunization. Mucosal immunization provides several potential advantages over subcutaneous and intramuscular routes, including protection from localized infection at the site of entry, clearance of organisms on mucosal surfaces, induction of long-term immunity through establishment of central and tissue-resident memory cells, and the ability to shape regulatory responses. Despite these advantages, significant barriers remain to achieving effective mucosal immunization. The epithelium itself provides many obstacles to immunization, and the activation of immune recognition and effector pathways that leads to mucosal immunity has been difficult to achieve. This review will highlight the potential advantages of mucosal immunity, define the barriers to mucosal immunization, examine the immune mechanisms that need to be activated on mucosal surfaces, and finally address recent developments in methods for mucosal vaccination that have shown promise in generating immunity on mucosal surfaces in human trials.

The Bacterial Spore as a Mucosal Vaccine Delivery System.

The development of efficient mucosal vaccines is strongly dependent on the use of appropriate vectors. Various biological systems or synthetic nanoparticles have been proposed to display and deliver antigens to mucosal surfaces. The Bacillus spore, a metabolically quiescent and extremely resistant cell, has also been proposed as a mucosal vaccine delivery system and shown able to conjugate the advantages of live and synthetic systems. Several antigens have been displayed on the spore by either recombinant or non-recombinant approaches, and antigen-specific immune responses have been observed in animals immunized by the oral or nasal route. Here we review the use of the bacterial spore as a mucosal vaccine vehicle focusing on the advantages and drawbacks of using the spore and of the recombinant vs. non-recombinant approach to display antigens on the spore surface. An overview of the immune responses induced by antigen-displaying spores so far tested in animals is presented and discussed.

Evaluation of Immune Response to Mucosal Immunization with an Oral Probiotic-Based Vaccine in Mice: Potential for Prime-Boost Immunization against SARS-CoV-2.

Following the conclusion of the COVID-19 pandemic, the persistent genetic variability in the virus and its ongoing circulation within the global population necessitate the enhancement of existing preventive vaccines and the development of novel ones. A while back, we engineered an orally administered probiotic-based vaccine, L3-SARS, by integrating a gene fragment that encodes the spike protein S of the SARS-CoV-2 virus into the genome of the probiotic strain E. faecium L3, inducing the expression of viral antigen on the surface of bacteria. Previous studies demonstrated the efficacy of this vaccine candidate in providing protection against the virus in Syrian hamsters. In this present study, utilizing laboratory mice, we assess the immune response subsequent to immunization via the gastrointestinal mucosa and discuss its potential as an initial phase in a two-stage vaccination strategy. Our findings indicate that the oral administration of L3-SARS elicits an adaptive immune response in mice. Pre-immunization with L3-SARS enhances and prolongs the humoral immune response following a single subcutaneous immunization with a recombinant S-protein analogous to the S-insert of the coronavirus in Enterococcus faecium L3.

Efficacy of mucosal vaccination using a protozoan parasite as a vehicle for antigen delivery: IgG and neutralizing response after rectal administration of LeCoVax-2, a candidate vaccine against COVID-19.

Mucosal vaccination is regarded as a promising alternative to classical, intramuscular vaccine delivery. However, only a limited number of vaccines have been licensed for mucosal administration in humans. Here we propose Leishmania tarentolae, a protozoan parasite, as a potential antigen vehicle for mucosal vaccination, for administration via the rectal or oral routes. To test this hypothesis, we exploited L. tarentolae for the production and delivery of SARS-CoV-2 antigens. Two antigens were assayed in BALB/c mice: Lt-spike, a L. tarentolae clone engineered for the surface expression of the SARS-CoV-2 spike protein; RBD-SD1, a purified portion of the spike protein, produced by another engineered clone of the protozoon. Immune response parameters were then determined at different time points. Both antigens, administered either separately or in combination (Lt-spike + RBD-SD1, hereafter LeCoVax-2), determined significant IgG seroconversion and production of neutralizing antibodies after subcutaneous administration, but only in the presence of adjuvants. After rectal administration, the purified RBD-SD1 antigen did not induce any detectable immune response, in comparison with the intense response observed after administration of LeCoVax-2 or Lt-spike alone. In rectal administration, LeCoVax-2 was also effective when administered without adjuvant. Our results show that L. tarentolae is an efficient and safe scaffold for production and delivery of viral antigens, to be used as vaccines. In addition, rectal vaccination experiments prove that L. tarentolae is suitable as a vaccine vehicle and adjuvant for enteral vaccination. Finally, the combined preparation LeCoVax-2 can be considered as a promising candidate vaccine against SARS-CoV-2, worthy of further investigation.

Intranasal Immunization with Acellular Pertussis Vaccines Results in Long-Term Immunity to Bordetella pertussis in Mice.

Bordetella pertussis colonizes the respiratory mucosa of humans, inducing an immune response seeded in the respiratory tract. An individual, once convalescent, exhibits long-term immunity to the pathogen. Current acellular pertussis (aP) vaccines do not induce the long-term immune response observed after natural infection in humans. In this study, we evaluated the durability of protection from intranasal (i.n.) pertussis vaccines in mice. Mice that convalesced from B. pertussis infection served as a control group. Mice were immunized with a mock vaccine (phosphate-buffered saline [PBS]), aP only, or an aP base vaccine combined with one of the following adjuvants: alum, curdlan, or purified whole glucan particles (IRI-1501). We utilized two study designs: short term (challenged 35 days after priming vaccination) and long term (challenged 6 months after boost). The short-term study demonstrated that immunization with i.n. vaccine candidates decreased the bacterial burden in the respiratory tract, reduced markers of inflammation, and induced significant serum and lung antibody titers. In the long-term study, protection from bacterial challenge mirrored the results observed in the short-term challenge study. Immunization with pertussis antigens alone was surprisingly protective in both models; however, the alum and IRI-1501 adjuvants induced significant B. pertussis-specific IgG antibodies in both the serum and lung and increased numbers of anti-B. pertussis IgG-secreting plasma cells in the bone marrow. Our data indicate that humoral responses induced by the i.n. vaccines correlated with protection, suggesting that long-term antibody responses can be protective.

A Parainfluenza Virus Vector Expressing the Respiratory Syncytial Virus (RSV) Prefusion F Protein Is More Effective than RSV for Boosting a Primary Immunization with RSV.

Live-attenuated pediatric vaccines for intranasal administration are being developed for human respiratory syncytial virus (RSV), an important worldwide pediatric respiratory pathogen that lacks a licensed vaccine or suitable antiviral drug. We evaluated a prime-boost strategy in which primary immunization with RSV was boosted by secondary immunization with RSV or with a chimeric recombinant bovine/human parainfluenza virus type 3 (rB/HPIV3) vector expressing the RSV fusion F protein. The vector-expressed F protein had been engineered (DS-Cav1 mutations) for increased stability in the highly immunogenic prefusion (pre-F) conformation, with or without replacement of its transmembrane and cytoplasmic tail domains with their counterparts from bovine parainfluenza virus type 3 (BPIV3) F protein to direct incorporation into the vector virion for increased immunogenicity. In hamsters that received a primary infection with RSV, a booster infection with RSV ∼6 weeks later was completely restricted for producing infectious virus but induced a significant increase in the serum RSV-plaque-reduction neutralizing antibody titer (RSV-PRNT). Boosting instead with the rB/HPIV3-RSV-pre-F vectors resulted in efficient replication and induced significantly higher RSV-PRNTs than RSV. In African green monkeys that received a primary infection with RSV, a booster infection with RSV ∼2, ∼6, or ∼15 months later was highly restricted, whereas booster infections with the vectors had robust replication. Compared with RSV, boosts with the vectors induced 7- to 15-fold higher titers of RSV-specific serum antibodies with high neutralizing activity, as well as significantly higher titers of RSV-specific mucosal IgA antibodies. These findings support further development of this heterologous prime-boost strategy.IMPORTANCE Immune responses to RSV in infants can be reduced due to immunological immaturity and immunosuppression by RSV-specific maternal antibodies. In infants and young children, two infections with wild-type RSV typically are needed to achieve the titers of RSV-specific serum antibodies and protection against illness that are observed in adults. Therefore, a boost might substantially improve the performance of live pediatric RSV vaccines presently being developed. Hamsters and African green monkeys received a primary intranasal infection with RSV and were given a boost with RSV or a parainfluenza virus (PIV) vector expressing RSV fusion protein engineered for enhanced immunogenicity. The RSV boost was highly restricted but induced a significant increase in serum RSV-neutralizing antibodies. The PIV vectors replicated efficiently and induced significantly higher antibody responses. The use of an attenuated PIV vector expressing RSV antigen to boost a primary immunization with an attenuated RSV warrants further evaluation.

Chlamydia-Specific IgA Secretion in the Female Reproductive Tract Induced via Per-Oral Immunization Confers Protection against Primary Chlamydia Challenge.

Chlamydia trachomatis is an obligate intracellular pathogen that causes sexually transmitted disease. In women, chlamydial infections may cause pelvic inflammatory disease (PID), ectopic pregnancy, and infertility. The role of antibodies in protection against a primary Chlamydia infection is unclear and was a focus of this work. Using the C. muridarum mouse infection model, we show that intestinal mucosa is infected via intranasal (i.n.) or per-oral (p.o.) Chlamydia inoculation and that unlike the female reproductive tract (FRT) mucosa, it halts systemic Chlamydia dissemination. Moreover, p.o. immunization or infection with Chlamydia confers protection against per-vaginal (p.v.) challenge, resulting in significantly decreased bacterial burden in the FRT, accelerated Chlamydia clearance, and reduced hydrosalpinx pathology. In contrast, subcutaneous (s.c.) immunization conferred no protection against the p.v. challenge. Both p.o. and s.c. immunizations induced Chlamydia-specific serum IgA. However, IgA was found only in the vaginal washes and fecal extracts of p.o.-immunized animals. Following a p.v. challenge, unimmunized control and s.c.-s.c.-immunized animals developed Chlamydia-specific intestinal IgA yet failed to develop IgA in the FRT, indicating that IgA response in the FRT relies on the FRT to gastrointestinal tract (GIT) antigen transport. Vaginal secretions of p.o.-immunized animals neutralize Chlamydia in vivo, resulting in significantly lower Chlamydia burden in the FRT and Chlamydia transport to the GIT. We also show that infection of the GIT is not necessary for induction of protective immunity in the FRT, a finding that is important for the development of p.o. subunit vaccines to target Chlamydia and possibly other sexually transmitted pathogens.

Optimized Mucosal Modified Vaccinia Virus Ankara Prime/Soluble gp120 Boost HIV Vaccination Regimen Induces Antibody Responses Similar to Those of an Intramuscular Regimen.

The benefits of mucosal vaccines over injected vaccines are difficult to ascertain, since mucosally administered vaccines often induce serum antibody responses of lower magnitude than those induced by injected vaccines. This study aimed to determine if mucosal vaccination using a modified vaccinia virus Ankara expressing human immunodeficiency virus type 1 (HIV-1) gp120 (MVAgp120) prime and a HIV-1 gp120 protein boost could be optimized to induce serum antibody responses similar to those induced by an intramuscularly (i.m.) administered MVAgp120 prime/gp120 boost to allow comparison of an i.m. immunization regimen to a mucosal vaccination regimen for the ability to protect against a low-dose rectal simian-human immunodeficiency virus (SHIV) challenge. A 3-fold higher antigen dose was required for intranasal (i.n.) immunization with gp120 to induce serum anti-gp120 IgG responses not significantly different than those induced by i.m. immunization. gp120 fused to the adenovirus type 2 fiber binding domain (gp120-Ad2F), a mucosal targeting ligand, exhibited enhanced i.n. immunogenicity compared to gp120. MVAgp120 was more immunogenic after i.n. delivery than after gastric or rectal delivery. Using these optimized vaccines, an i.n. MVAgp120 prime/combined i.m. (gp120) and i.n. (gp120-Ad2F) boost regimen (i.n./i.m.-plus-i.n.) induced serum anti-gp120 antibody titers similar to those induced by the intramuscular prime/boost regimen (i.m./i.m.) in rabbits and nonhuman primates. Despite the induction of similar systemic anti-HIV-1 antibody responses, neither the i.m./i.m. nor the i.n./i.m.-plus-i.n. regimen protected against a repeated low-dose rectal SHIV challenge. These results demonstrate that immunization regimens utilizing the i.n. route are able to induce serum antigen-specific antibody responses similar to those induced by systemic immunization.IMPORTANCE Mucosal vaccination is proposed as a method of immunization able to induce protection against mucosal pathogens that is superior to protection provided by parenteral immunization. However, mucosal vaccination often induces serum antigen-specific immune responses of lower magnitude than those induced by parenteral immunization, making the comparison of mucosal and parenteral immunization difficult. We identified vaccine parameters that allowed an immunization regimen consisting of an i.n. prime followed by boosters administered by both i.n. and i.m. routes to induce serum antibody responses similar to those induced by i.m. prime/boost vaccination. Additional studies are needed to determine the potential benefit of mucosal immunization for HIV-1 and other mucosally transmitted pathogens.

Effects of Alterations to the CX3C Motif and Secreted Form of Human Respiratory Syncytial Virus (RSV) G Protein on Immune Responses to a Parainfluenza Virus Vector Expressing the RSV G Protein.

Human respiratory syncytial virus (RSV) is a major pediatric respiratory pathogen. The attachment (G) and fusion (F) glycoproteins are major neutralization and protective antigens. RSV G is expressed as membrane-anchored (mG) and -secreted (sG) forms, both containing a central fractalkine-like CX3C motif. The CX3C motif and sG are thought to interfere with host immune responses and have been suggested to be omitted from a vaccine. We used a chimeric bovine/human parainfluenza virus type 3 (rB/HPIV3) vector to express RSV wild-type (wt) G and modified forms, including sG alone, mG alone, mutants with ablated CX3C, and G with enhanced packaging into vector virions. In hamsters, these viruses replicated to similar titers. When assayed with a complement-enhanced neutralization assay in Vero cells, sG did not reduce the serum RSV- or PIV3-neutralizing antibody (NAb) responses, whereas ablating CX3C drastically reduced the RSV NAb response. Protective efficacy against RSV challenge was not reduced by sG but was strongly dependent on the CX3C motif. In ciliated human airway epithelial (HAE) cells, NAbs induced by wt G, but not by wt F, completely blocked RSV infection in the absence of added complement. This activity was dependent on the integrity of the CX3C motif. In hamsters, the rB/HPIV3 expressing wt G conferred better protection against RSV challenge than that expressing wt F. Codon optimization of the wt G further increased its immunogenicity and protective efficacy. This study showed that ablation of the CX3C motif or sG in an RSV vaccine, as has been suggested previously, would be ill advised.IMPORTANCE Human RSV is the leading viral cause of severe pediatric respiratory illness. An RSV vaccine is not yet available. The RSV attachment protein G is an important protective and neutralization antigen. G contains a conserved fractalkine-like CX3C motif and is expressed in mG and sG forms. sG and the CX3C motif are thought to interfere with host immune responses, but this remains poorly characterized. Here, we used an attenuated chimeric bovine/human parainfluenza virus type 3 (rB/HPIV3) vector to express various modified forms of RSV G. We demonstrated that strong antibody and protective responses could be induced by G alone, and that this was highly dependent on the integrity of the CX3C motif. There was no evidence that sG or the CX3C motif impaired immune responses against RSV G or the rB/HPIV3 vector. rB/HPIV3 expressing wt RSV G provides a bivalent vaccine against RSV and HPIV3.

The temperature of growth and sporulation modulates the efficiency of spore-display in Bacillus subtilis.

BACKGROUND: Bacterial spores displaying heterologous antigens or enzymes have long been proposed as mucosal vaccines, functionalized probiotics or biocatalysts. Two main strategies have been developed to display heterologous molecules on the surface of Bacillus subtilis spores: (i) a recombinant approach, based on the construction of a gene fusion between a gene coding for a coat protein (carrier) and DNA coding for the protein to be displayed, and (ii) a non-recombinant approach, based on the spontaneous and stable adsorption of heterologous molecules on the spore surface. Both systems have advantages and drawbacks and the selection of one or the other depends on the protein to be displayed and on the final use of the activated spore. It has been recently shown that B. subtilis builds structurally and functionally different spores when grown at different temperatures; based on this finding B. subtilis spores prepared at 25, 37 or 42 °C were compared for their efficiency in displaying various model proteins by either the recombinant or the non-recombinant approach. RESULTS: Immune- and fluorescence-based assays were used to analyze the display of several model proteins on spores prepared at 25, 37 or 42 °C. Recombinant spores displayed different amounts of the same fusion protein in response to the temperature of spore production. In spores simultaneously displaying two fusion proteins, each of them was differentially displayed at the various temperatures. The display by the non-recombinant approach was only modestly affected by the temperature of spore production, with spores prepared at 37 or 42 °C slightly more efficient than 25 °C spores in adsorbing at least some of the model proteins tested. CONCLUSION: Our results indicate that the temperature of spore production allows control of the display of heterologous proteins on spores and, therefore, that the spore-display strategy can be optimized for the specific final use of the activated spores by selecting the display approach, the carrier protein and the temperature of spore production.

Progress, challenges and opportunities in fish vaccine development.

In 2014 the contribution of aquaculture to supply food for human consumption overtook wild-caught fish for the first time. Despite improvements in the aquaculture industry, it has been estimated that as much as 10% of all cultured aquatic animals are lost because of infectious diseases, amounting to >10 billion USD in losses annually on a global scale. Vaccination to prevent disease is used routinely in finfish aquaculture, especially for Atlantic salmon (Salmo salar), while in a limited capacity (or not at all) in many other fish species due to lack of vaccines, poor performance or cost. There has, nevertheless, been impressive progress in fish vaccine development over the last 4 decades with 24 licenced fish vaccines now commercially available for use in a variety of fish species. These comprise whole killed, peptide subunit, recombinant protein, DNA and live attenuated vaccines. Challenges do, however, still exist as the majority of commercial vaccines are killed whole cell pathogen preparations administered by intraperitoneal injection. This may not be the optimal route to deliver some vaccines, but lack of effective adjuvants and basic knowledge on immune response has hindered progress in the development of mucosal vaccines. The cost of injecting fish may also be prohibitive in some countries leading to disease treatment (e.g. with antibiotics) rather than using preventative measures. It is important that these issues are addressed as the industry continues to grow globally. Exciting opportunities exist for rapid development of fish vaccines in the future, with continued reduction in cost of technologies (e.g. of whole genome sequencing), regulations changing (e.g. DNA vaccines can now authorised in Europe), the introduction of novel antigen expression and delivery systems (such as virus-like particles, VLPs), development of novel adjuvants and advancements in the elucidation of basic mechanisms of mucosal immunity. Development of effective mucosal vaccines and optimisation of their delivery will facilitate novel vaccine development, and enable the aquaculture industries in LMIC to use vaccination routinely in the future. In addition, effective use of emergency (autogenous) vaccines will assist in tackling emerging disease challenges.

Attenuated Human Parainfluenza Virus Type 1 Expressing the Respiratory Syncytial Virus (RSV) Fusion (F) Glycoprotein from an Added Gene: Effects of Prefusion Stabilization and Packaging of RSV F.

Human respiratory syncytial virus (RSV) is the most prevalent worldwide cause of severe respiratory tract infection in infants and young children. Human parainfluenza virus type 1 (HPIV1) also causes severe pediatric respiratory illness, especially croup. Both viruses lack vaccines. Here, we describe the preclinical development of a bivalent RSV/HPIV1 vaccine based on a recombinant HPIV1 vector, attenuated by a stabilized mutation, that expresses RSV F protein modified for increased stability in the prefusion (pre-F) conformation by previously described disulfide bond (DS) and hydrophobic cavity-filling (Cav1) mutations. RSV F was expressed from the first or second gene position as the full-length protein or as a chimeric protein with its transmembrane and cytoplasmic tail (TMCT) domains substituted with those of HPIV1 F in an effort to direct packaging in the vector particles. All constructs were recovered by reverse genetics. The TMCT versions of RSV F were packaged in the rHPIV1 particles much more efficiently than their full-length counterparts. In hamsters, the presence of the RSV F gene, and in particular the TMCT versions, was attenuating and resulted in reduced immunogenicity. However, the vector expressing full-length RSV F from the pre-N position was immunogenic for RSV and HPIV1. It conferred complement-independent high-quality RSV-neutralizing antibodies at titers similar to those of wild-type RSV and provided protection against RSV challenge. The vectors exhibited stable RSV F expression in vitro and in vivo In conclusion, an attenuated rHPIV1 vector expressing a pre-F-stabilized form of RSV F demonstrated promising immunogenicity and should be further developed as an intranasal pediatric vaccine.IMPORTANCE RSV and HPIV1 are major viral causes of acute pediatric respiratory illness for which no vaccines or suitable antiviral drugs are available. The RSV F glycoprotein is the major RSV neutralization antigen. We used a rHPIV1 vector, bearing a stabilized attenuating mutation, to express the RSV F glycoprotein bearing amino acid substitutions that increase its stability in the pre-F form, the most immunogenic form that elicits highly functional virus-neutralizing antibodies. RSV F was expressed from the pre-N or N-P gene position of the rHPIV1 vector as a full-length protein or as a chimeric form with its TMCT domain derived from HPIV1 F. TMCT modification greatly increased packaging of RSV F into the vector particles but also increased vector attenuation in vivo, resulting in reduced immunogenicity. In contrast, full-length RSV F expressed from the pre-N position was immunogenic, eliciting complement-independent RSV-neutralizing antibodies and providing protection against RSV challenge.

Immunity Provided by an Outer Membrane Vesicle Cholera Vaccine Is Due to O-Antigen-Specific Antibodies Inhibiting Bacterial Motility.

An outer membrane vesicle (OMV)-based cholera vaccine is highly efficacious in preventing intestinal colonization in the suckling mouse model. Immunity from OMVs comes from immunoglobulin (Ig), particularly IgG, in the milk of mucosally immunized dams. Anti-OMV IgG renders Vibrio cholerae organisms immotile, thus they pass through the small intestine without colonizing. However, the importance of motility inhibition for protection and the mechanism by which motility is inhibited remain unclear. By using both in vitro and in vivo experiments, we found that IgG inhibits motility by specifically binding to the O-antigen of V. cholerae We demonstrate that the bivalent structure of IgG, although not required for binding to the O-antigen, is required for motility inhibition. Finally, we show using competition assays in suckling mice that inhibition of motility appears to be responsible for most, if not all, of the protection engendered by OMV vaccination, thus providing insight into the mechanism of immune protection.

Lactic acid bacteria as mucosal delivery vehicles: a realistic therapeutic option.

Recombinant lactic acid bacteria (LAB), in particular lactococci and lactobacilli, have gained increasing interest as mucosal delivery vehicles in recent years. With the development of mucosal vaccines, studies on LAB expression systems have been mainly focused on the generation of genetic tools for antigen expression in different locations. Recombinant LAB show advantages in a wide range of aspects over other mucosal delivery systems and represent an attractive candidate for the delivery of therapeutic and prophylactic molecules in different applications. Here, we review the recent data on the use of recombinant LAB as mucosal delivery vectors and the associated health benefits, including the prevention and treatment of inflammatory bowel diseases (IBDs), autoimmune disorders, and infections by pathogenic microorganisms from mucosal surfaces. In addition, we discuss the use of LAB as vehicles to deliver DNA directly to eukaryotic cells. Researches from the last 5 years demonstrate that LAB as vectors for mucosal delivery of therapeutic molecules seem to be a realistic therapeutic option both in human and animal diseases.

Protective effect of intranasal immunization with Neospora caninum membrane antigens against murine neosporosis established through the gastrointestinal tract.

Neospora caninum is an Apicomplexa parasite that in the last two decades was acknowledged as the main pathogenic agent responsible for economic losses in the cattle industry. In the present study, the effectiveness of intranasal immunization with N. caninum membrane antigens plus CpG adjuvant was assessed in a murine model of intragastrically established neosporosis. Immunized mice presented a lower parasitic burden in the brain on infection with 5 × 10(7) tachyzoites, showing that significant protection was achieved by this immunization strategy. Intestinal IgA antibodies raised by immunization markedly agglutinated live N. caninum tachyzoites whereas previous opsonization with IgG antibodies purified from immunized mice sera reduced parasite survival within macrophage cells. Although an IgG1 : IgG2a ratio < 1 was detected in the immunized mice before and after infection, indicative of a predominant T helper type 1 immune response, no increased production of interferon-γ was detected in the spleen or mesenteric lymph nodes of the immunized mice. Altogether, these results show that mucosal immunization with N. caninum membrane proteins plus CpG adjuvant protect against intragastrically established neosporosis and indicate that parasite-specific mucosal and circulating antibodies have a protective role against this parasitic infection.