Intranasal Immunization Of Mice Research Articles

Display of FliC131 on the Surface of Lactococcus lactis as a Strategy to Increase its Adjuvanticity for Mucosal Immunization

Research on innovative mucosal adjuvants is essential to develop new vaccines for safe mucosal application. In this work, we propose the development of a Lactococcus lactis that expresses a variant of flagellin on its surface (FliC131*), to increase the adjuvanticity of the living cell and cell wall-derived particles (CWDP). We optimized the expression of FliC131*, and confirmed its identity and localization by Western blot and flow cytometry. We also generated CWDP containing FliC131* (CDWP-FliC131*) and evaluated their storage stability. Lastly, we measured the human TLR5 stimulating activity in vitro and assessed the adjuvanticity in vivo using ovalbumin (OVA) as a model antigen. As a result, we generated L. lactis/pCWA-FliC131*, that expresses and displays FliC131* on its surface, obtained the corresponding CWDP-FliC131*, and showed that both activated hTLR5 in vitro in a dose-dependent manner. Furthermore, CWDP-FliC131* retained this biological activity after being lyophilized and stored for a year. Finally, intranasal immunization of mice with OVA plus live L. lactis/pCWA-FliC131* or CWDP-FliC131* induced OVA-specific IgG and IgA in serum, intestinal lavages, and bronchoalveolar lavages. Our work demonstrates the potential of this recombinant L. lactis with an enhanced adjuvant effect, prompting its further evaluation for the design of novel mucosal vaccines.

Intranasal immunization of mice with chimera of Salmonella Typhi protein elicits protective intestinal immunity

Development of safe, highly effective and affordable enteric fever vaccines is a global health priority. Live, oral typhoid vaccines induce strong mucosal immunity and long-term protection, but safety remains a concern. In contrast, efficacy wears off rapidly for injectable, polysaccharide-based vaccines, which elicit poor mucosal response. We previously reported Salmonella Typhi outer membrane protein, T2544 as a potential candidate for bivalent (S. Typhi and S. Paratyphi A) vaccine development. Here, we show that intranasal immunization with a subunit vaccine (chimera of T2544 and cholera toxin B subunit) induced strong systemic and intestinal mucosal immunity and protection from S. Typhi challenge in a mouse model. CTB-T2544 augmented gut-homing receptor expression on lymphocytes that produced Th1 and Th17 cytokines, secretory IgA in stool that inhibited bacterial motility and epithelial attachment, antibody recall response and affinity maturation with increased number of follicular helper T cells and CD4+ central and effector memory cells.

SARS-CoV-2 spike-FLIPr fusion protein plus lipidated FLIPr protects against various SARS-CoV-2 variants in hamsters.

Vaccine-induced mucosal immunity and broad protective capacity against various severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants remain inadequate. Formyl peptide receptor-like 1 inhibitory protein (FLIPr), produced by Staphylococcus aureus, can bind to various Fcγ receptor subclasses. Recombinant lipidated FLIPr (rLF) was previously found to be an effective adjuvant. In this study, we developed a vaccine candidate, the recombinant Delta SARS-CoV-2 spike (rDS)-FLIPr fusion protein (rDS-F), which employs the property of FLIPr binding to various Fcγ receptors. Our study shows that rDS-F plus rLF promotes rDS capture by dendritic cells. Intranasal vaccination of mice with rDS-F plus rLF increases persistent systemic and mucosal antibody responses and CD4/CD8 T-cell responses. Importantly, antibodies induced by rDS-F plus rLF vaccination neutralize Delta, Wuhan, Alpha, Beta, and Omicron strains. Additionally, rDS-F plus rLF provides protective effects against various SARS-CoV-2 variants in hamsters by reducing inflammation and viral loads in the lung. Therefore, rDS-F plus rLF is a potential vaccine candidate to induce broad protective responses against various SARS-CoV-2 variants.IMPORTANCEMucosal immunity is vital for combating pathogens, especially in the context of respiratory diseases like COVID-19. Despite this, most approved vaccines are administered via injection, providing systemic but limited mucosal protection. Developing vaccines that stimulate both mucosal and systemic immunity to address future coronavirus mutations is a growing trend. However, eliciting strong mucosal immune responses without adjuvants remains a challenge. In our study, we have demonstrated that using a recombinant severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike-formyl peptide receptor-like 1 inhibitory protein (FLIPr) fusion protein as an antigen, in combination with recombinant lipidated FLIPr as an effective adjuvant, induced simultaneous systemic and mucosal immune responses through intranasal immunization in mice and hamster models. This approach offered protection against various SARS-CoV-2 strains, making it a promising vaccine candidate for broad protection. This finding is pivotal for future broad-spectrum vaccine development.

An FcRn-targeted mucosal vaccine against SARS-CoV-2 infection and transmission

SARS-CoV-2 is primarily transmitted through droplets and airborne aerosols, and in order to prevent infection and reduce viral spread vaccines should elicit protective immunity in the airways. The neonatal Fc receptor (FcRn) transfers IgG across epithelial barriers and can enhance mucosal delivery of antigens. Here we explore FcRn-mediated respiratory delivery of SARS-CoV-2 spike (S). A monomeric IgG Fc was fused to a stabilized spike; the resulting S-Fc bound to S-specific antibodies and FcRn. Intranasal immunization of mice with S-Fc and CpG significantly induced antibody responses compared to the vaccination with S alone or PBS. Furthermore, we intranasally immunized mice or hamsters with S-Fc. A significant reduction of virus replication in nasal turbinate, lung, and brain was observed following nasal challenges with SARS-CoV-2 and its variants. Intranasal immunization also significantly reduced viral airborne transmission in hamsters. Nasal IgA, neutralizing antibodies, lung-resident memory T cells, and bone-marrow S-specific plasma cells mediated protection. Hence, FcRn delivers an S-Fc antigen effectively into the airway and induces protection against SARS-CoV-2 infection and transmission.

A bivalent outer membrane vesicle-based intranasal vaccine to prevent infection of periodontopathic bacteria

Periodontal disease has become a serious public health problem, not only causing tooth loss, but also inducing chronic disorders of extra-oral organs. The present study assessed an intranasal vaccine strategy to prevent periodontal disease using outer membrane vesicles (OMVs) of two major periodontopathic bacteria, Porphyromonas gingivalis (Pg) and Aggregatibacter actinomycetemcomitans (Aa). We compared the morphology, composition, and immune activity between OMVs of Pg strain ATCC 33277 and Aa strain Y4. Aa OMVs had a smoother surface and stronger lipid A activity compared to Pg OMVs. The in vitro immune activity elicited by Aa OMVs in macrophage-like cells was remarkably stronger than that of Pg OMVs. Intranasal immunization of mice with Aa OMVs alone resulted in robust, humoral immune responses in blood and saliva. Despites the intrinsically low mucosal immunogenicity of Pg OMVs alone, using Aa OMVs as a mucosal adjuvant strongly enhanced Pg-specific immune responses, resulting in both serum IgG and salivary IgA, both of which aggregated Pg and Aa cells. Furthermore, Aa OMVs were found to be a more potent mucosal adjuvant than Poly(I:C) in the context of enhancing the production of Pg-specific IgG (especially IgG2a) and IgA. In addition, in a randomized, blinded study, mice oral challenged with Pg and Aa after intranasal immunization with Pg OMVs and Aa OMVs had significantly decreased numbers of both microorganisms compared to mock-immunized mice. Furthermore, in an intracerebral injection mouse model, there were no serious adverse effects on the brain even after administrating a dose of OMVs as same as that used for intranasal administration. Taken together, the bivalent OMV intranasal vaccine may be effective in preventing colonization of periodontopathic bacteria in the oral cavity and related systemic disorders associated with periodontal diseases.

Mucosal immunization with lipopeptides derived from conserved regions of SARS-CoV-2 antigens induce robust cellular and cross-variant humoral immune responses in mice.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, has infected >600 million people in the ongoing global pandemic. Several variants of the SARS-CoV-2 have emerged in the last >2 years, challenging the continued efficacy of current COVID vaccines. Therefore, there is a crucial need to investigate a highly cross-protective vaccine effective against variants of SARS-CoV-2. In this study, we examined seven lipopeptides derived from highly conserved, immunodominant epitopes from the S, N, and M proteins of SARS-CoV-2, that are predicted to contain epitopes for clinically protective B cells, helper T cells (TH) and cytotoxic T cells (CTL). Intranasal immunization of mice with most of the lipopeptides led to significantly higher splenocyte proliferation and cytokine production, mucosal and systemic antibody responses, and induction of effector B and T lymphocytes in both lungs and spleen, compared to immunizations with the corresponding peptides without lipid. Immunizations with Spike-derived lipopeptides led to cross-reactive IgG, IgM and IgA responses against Alpha, Beta, Delta, and Omicron Spike proteins as well as neutralizing antibodies. These studies support their potential for development as components of a cross-protective SARS-CoV-2 vaccine.

Bystander activation of Bordetella pertussis-induced nasal tissue-resident memory CD4 T cells confers heterologous immunity to Klebsiella pneumoniae.

Tissue-resident memory CD4 T (TRM ) cells induced by infection with Bordetella pertussis persist in respiratory tissues and confer long-term protective immunity against reinfection. However, it is not clear how they are maintained in respiratory tissues. Here, we demonstrate that B. pertussis-specific CD4 TRM cells produce IL-17A in response to in vitro stimulation with LPS or heat-killed Klebsiella pneumoniae (HKKP) in the presence of dendritic cells. Furthermore, IL-17A-secreting CD4 TRM cells expand in the lung and nasal tissue of B. pertussis convalescent mice following in vivo administration of LPS or HKKP. Bystander activation of CD4 TRM cells was suppressed by anti-IL-12p40 but not by anti-MHCII antibodies. Furthermore, purified respiratory tissue-resident, but not circulating, CD4 T cells from convalescent mice produced IL-17A following direct stimulation with IL-23 and IL-1β or IL-18. Intranasal immunization of mice with a whole-cell pertussis vaccine induced respiratory CD4 TRM cells that were reactivated following stimulation with K. pneumoniae. Furthermore, the nasal pertussis vaccine conferred protective immunity against B. pertussis but also attenuated infection with K. pneumoniae. Our findings demonstrate that CD4 TRM cells induced by respiratory infection or vaccination can undergo bystander activation and confer heterologous immunity to an unrelated respiratory pathogen.

Local and systemic immune responses induced by intranasal immunization with biomineralized foot-and-mouth disease virus-like particles.

Foot-and-mouth disease virus (FMDV) infects the host by invading mucosal epithelial cells of the respiratory or digestive tract. Therefore, establishing a specific antiviral mucosal immune barrier can effectively block viral invasion. We evaluated local mucosal and systemic immune responses elicited by intranasal immunization of mice with foot-and-mouth disease (FMD) calcium phosphate mineralized virus-like particles (CaP-VLPs) and tested whether three commercial mucosal adjuvants enhanced the immunogenicity of the antigen. The biosafety of the vaccine was verified through gross observation and pathological analysis of the lungs. CaP-VLPs effectively induced secretion of IgA (sIgA) from multiple sites in mouse mucosa and produced anti-FMD-specific IgG in the serum. Splenic lymphocytes specifically proliferated and secreted IFN-γ following antigen stimulation, indicating the vaccine can induce a certain level of cellular immune response. Finally, the pathological examination confirmed that CaP-VLPs did not cause substantial damage to the lungs of animals after immunization via mucosal administration. Notably, the vaccine mixed with S adjuvant increased the content of sIgA and serum IgG, and the high level of IgG in serum was maintained at least 7 weeks. Overall, this study reveals that FMD CaP-VLPs can induce good local mucosal immune and systemic immune response through intranasal immunization, and the immune response was specifically enhanced by S adjuvant. These data support that CaP-VLPs-S as a candidate mucosal vaccine for the prevention of FMD vaccine infection.

Enhancement of mucosal innate and adaptive immunity following intranasal immunization of mice with a bovine adenoviral vector.

Nonhuman adenoviral (AdV) gene delivery platforms have significant value due to their ability to elude preexisting AdV vector immunity in most individuals. Previously, we have demonstrated that intranasal (IN) immunization of mice with BAd-H5HA, a bovine AdV type 3 (BAdV3) vector expressing H5N1 influenza virus hemagglutinin (HA), resulted in enhanced humoral and cell-mediated immune responses. The BAd-H5HA IN immunization resulted in complete protection following the challenge with an antigenically distinct H5N1 virus compared to the mouse group similarly immunized with HAd-H5HA, a human AdV type 5 (HAdV5) vector expressing HA. Here, we attempted to determine the activation of innate immune responses in the lungs of mice inoculated intranasally with BAd-H5HA compared to the HAd-H5HA-inoculated group. RNA-Seq analyses of the lung tissues revealed differential expression (DE) of genes involved in innate and adaptive immunity in animals immunized with BAd-H5HA. The top ten enhanced genes were verified by RT-PCR. Consistently, there were transient increases in the levels of cytokines (IL-1α, IL-1β, IL-5, TNF- α, LIF, IL-17, G-CSF, MIP-1β, MCP-1, MIP-2, and GM-CSF) and toll-like receptors in the lungs of the group inoculated with BAdV vectors compared to that of the HAdV vector group. These results demonstrate that the BAdV vectors induce enhanced innate and adaptive immunity-related factors compared to HAdV vectors in mice. Thus, the BAdV vector platform could be an excellent gene delivery system for recombinant vaccines and cancer immunotherapy.

Associated virus-bacterial vaccine based on seasonal LAIV and S. pneumoniae chimeric peptide provide protection against post-influenza pneumococcal infection in mouse model

ABSTRACT Severe influenza complications are often caused by Streptococcus pneumoniae infection, which presents the most common cause of community-acquired pneumonia. We evaluated in a mouse model an associated virus-bacterial vaccine based on seasonal live influenza vaccines (LAIV) and S. pneumoniae chimeric protein comprising flagellin (PSPF). Intranasal immunization of mice with a complex of trivalent LAIV and PSPF caused an increased release of early cytokines in the lungs of mice. The immunogenicity of LAIV and PSPF in the associated vaccine composition was sometimes decreased compared to each vaccine preparation alone. Nevertheless, only vaccination of mice with LAIV+PSPF significantly reduced lethality and the bacterial load in the lungs in a model of post-influenza bacterial pneumonia. The study of the interactions of influenza viruses with bacterial peptides is important during the development of associated virus-bacterial vaccines intended for the prevention of severe post-influenza bacterial complications.

Self-Assembly of Flagellin into Immunostimulatory Ring-like Nanostructures as an Antigen Delivery System.

Proteinaceous nanoparticles represent attractive antigen carriers for vaccination as their size and repetitive antigen displays that mimic most viral particles enable efficient immune processing. However, these nanocarriers are often unable to stimulate efficiently the innate immune system, requiring coadministration with adjuvants to promote long-lasting protective immunity. The protein flagellin, which constitutes the primary constituent of the bacterial flagellum, has been widely evaluated as an antigen carrier due to its intrinsic adjuvant properties involving activation of the innate immune receptor Toll-like receptor 5 (TLR5). Although flagellin is known for its ability to self-assemble into micron-scale length nanotubes, few studies have evaluated the potential usage of flagellin-based nanostructures as immunostimulatory antigen carriers. In this study, we reported for the first time a strategy to guide the self-assembly of a flagellin protein from Bacillus subtilis, Hag, into lower aspect ratio nanoparticles by hindering non-covalent interactions responsible for its elongation into nanotubes. We observed that addition of an antigenic sequence derived from the influenza A virus (3M2e) at the C-terminus of this flagellin, as opposed to positioning the epitope into mid-sequence, precluded filament elongation and resulted in low aspect ratio ring-like nanostructures upon salting-out-induced self-assembly. These nanostructures displayed the antigen at their surface and shared morphological and structural characteristics with flagellin nanotubes, with a diameter of approximately 12 nm, and an α-helix-rich secondary structure. Flagellin ring-like nanostructures were efficiently internalized by antigen-presenting cells, and avidly activated the TLR5 in vitro as well as the innate and adaptive immune responses. Intranasal immunization of mice with these nanostructures resulted in the potentiation of the antigen-specific antibody response and protection against a lethal infection with the influenza A virus, illustrating the potential of these intrinsically immunostimulatory nanostructures as antigen carriers.

Nanoparticles based on artificial self-assembling peptide and displaying M2e peptide and stalk HA epitopes of influenza A virus induce potent humoral and T-cell responses and protect against the viral infection

The extracellular domain of the M2 protein (M2e) and conserved region of the second subunit of the hemagglutinin (HA2) could be used for the development of broad-spectrum vaccines against influenza A. Here we obtained and characterized recombinant mosaic proteins containing tandem copies of M2e and HA2 fused to an artificial self-assembling peptide (SAP). The inclusion of SAP peptides in the fusion proteins enabled their self-assembly in vitro into spherical particles with a size of 30-50 nm. Intranasal immunization of mice with these particles without additional adjuvants induced strong humoral immune response against M2e and the whole virus. Particles carrying both M2e and HA2 induced antigen-specific multifunctional CD4+ effector memory T cells. Immunization provided high protection of mice against the lethal challenge with different subtypes of influenza A virus. The obtained self-assembling nanoparticles can be used to develop a universal influenza vaccine.

Mucosal Influenza Vector Vaccine Carrying TB10.4 and HspX Antigens Provides Protection against Mycobacterium tuberculosis in Mice and Guinea Pigs.

New strategies providing protection against tuberculosis (TB) are still pending. The airborne nature of Mycobacterium tuberculosis (M.tb) infection assumes that the mucosal delivery of the TB vaccine could be a more promising strategy than the systemic route of immunization. We developed a mucosal TB vaccine candidate based on recombinant attenuated influenza vector (Flu/THSP) co-expressing truncated NS1 protein NS1(1–124) and a full-length TB10.4 and HspX proteins of M.tb within an NS1 protein open reading frame. The Flu/THSP vector was safe and stimulated a systemic TB-specific CD4+ and CD8+ T-cell immune response after intranasal immunization in mice. Double intranasal immunization with the Flu/THSP vector induced protection against two virulent M.tb strains equal to the effect of BCG subcutaneous injection in mice. In a guinea pig TB model, one intranasal immunization with Flu/THSP improved protection against M.tb when tested as a vaccine candidate for boosting BCG-primed immunity. Importantly, enhanced protection provided by a heterologous BCG-prime → Flu/THSP vector boost immunization scheme was associated with a significantly reduced lung and spleen bacterial burden (mean decrease of 0.77 lg CFU and 0.72 lg CFU, respectively) and improved lung pathology 8.5 weeks post-infection with virulent M.tb strain H37Rv.

Immunization with SP_1992 (DiiA) Protein of Streptococcus pneumoniae Reduces Nasopharyngeal Colonization and Protects against Invasive Disease in Mice.

Knowledge-based vaccinology can reveal uncharacterized antigen candidates for a new generation of protein-based anti-pneumococcal vaccines. DiiA, encoded by the sp_1992 locus, is a surface protein containing either one or two repeats of a 37mer N-terminal motif that exhibits low interstrain variability. DiiA belongs to the core proteome, contains several conserved B-cell epitopes, and is associated with colonization and pathogenesis. Immunization with DiiA protein via the intraperitoneal route induced a strong IgG response, including different IgG subtypes. Vaccination with DiiA increased bacterial clearance and induced protection against sepsis, conferring 70% increased survival at 48 h post-infection when compared to the adjuvant control. The immunogenic response and survival rates in mice immunized with a truncated DiiA version lacking 119 N-terminal residues were remarkably lower, confirming the relevance of the repeat zone in the immunoprotection by DiiA. Intranasal immunization of mice with the entire recombinant protein elicited mucosal IgG and IgA responses that reduced bacterial colonization of the nasopharynx, confirming that this protein might be a vaccine candidate for reducing the carrier rate. DiiA constitutes an example of how functionally unannotated proteins may still represent promising candidates that can be used in prophylactic strategies against the pneumococcal carrier state and invasive disease.

Adhesins of Brucella: Their Roles in the Interaction with the Host.

A central aspect of Brucella pathogenicity is its ability to invade, survive, and replicate in diverse phagocytic and non-phagocytic cell types, leading to chronic infections and chronic inflammatory phenomena. Adhesion to the target cell is a critical first step in the invasion process. Several Brucella adhesins have been shown to mediate adhesion to cells, extracellular matrix components (ECM), or both. These include the sialic acid-binding proteins SP29 and SP41 (binding to erythrocytes and epithelial cells, respectively), the BigA and BigB proteins that contain an Ig-like domain (binding to cell adhesion molecules in epithelial cells), the monomeric autotransporters BmaA, BmaB, and BmaC (binding to ECM components, epithelial cells, osteoblasts, synoviocytes, and trophoblasts), the trimeric autotransporters BtaE and BtaF (binding to ECM components and epithelial cells) and Bp26 (binding to ECM components). An in vivo role has also been shown for the trimeric autotransporters, as deletion mutants display decreased colonization after oral and/or respiratory infection in mice, and it has also been suggested for BigA and BigB. Several adhesins have shown unipolar localization, suggesting that Brucella would express an adhesive pole. Adhesin-based vaccines may be useful to prevent brucellosis, as intranasal immunization in mice with BtaF conferred high levels of protection against oral challenge with B. suis.

Intranasal Immunization of Mice with Multiepitope Chimeric Vaccine Candidate Based on Conserved Autotransporters SigA, Pic and Sap, Confers Protection against Shigella flexneri.

Shigellosis is a diarrheal disease and the World Health Organization prompts the development of a vaccine against Shigella flexneri. The autotransporters SigA, Pic and Sap are conserved among Shigella spp. We previously designed an in silico vaccine with immunodominat epitopes from those autotransporters, and the GroEL protein of S. typhi as an adjuvant. Here, we evaluated the immunogenicity and protective efficacy of the chimeric multiepitope protein, named rMESF, in mice against lethal infection with S. flexneri. rMESF was administered to mice alone through the intranasal (i.n.) route or accompanied with Complete Freund’s adjuvant (CFA) intradermically (i.d.), subcutaneously (s.c.), and intramuscular (i.m.), as well as with Imject alum (i.m.). All immunized mice increased IgG, IgG1, IgG2a, IgA and fecal IgA titers compared to PBS+CFA and PBS+alum control groups. Furthermore, i.n. immunization of mice with rMESF alone presented the highest titers of serum and fecal IgA. Cytokine levels (IFN-γ, TNF-α, IL-4, and IL-17) and lymphocyte proliferation increased in all experimental groups, with the highest lymphoproliferative response in i.n. mice immunized with rMESF alone, which presented 100% protection against S. flexneri. In summary, this vaccine vests protective immunity and highlights the importance of mucosal immunity activation for the elimination of S. flexneri.

Intranasal delivery of cationic liposome-protamine complex mRNA vaccine elicits effective anti-tumor immunity

Immunization with synthetic mRNA encoding tumor-associated antigens is an emerging vaccine strategy for the treatment of cancer. In order to prevent mRNA degradation, promote antigen-presenting cells antigen presentation, and induce an anti-tumor immune response, we investigated the nasal administration of mRNA vaccines with positively charged protamine to concentrate mRNA, form a stable polycation-mRNA complex, and encapsulate the complex with DOTAP/Chol/DSPE-PEG cationic liposomes. Cationic liposome/protamine complex (LPC) showed significantly greater efficiency in uptake of vaccine particles in vitro and stronger capacities to stimulate dendritic cell maturation, which further induced a potent anti-tumor immune response. Intranasal immunization of mice with cationic LPC containing mRNA encoding cytokeratin 19 provoked a strong cellular immune response and slowed tumor growth in an aggressive Lewis lung cancer model. The results of this study provide evidence that cationic LPC can be used as a safe and effective adjuvant and this mRNA formulation provides a basis for anti-cancer vaccination of humans.

MERS-CoV Spike Protein Vaccine and Inactivated Influenza Vaccine Formulated with Single Strand RNA Adjuvant Induce T-Cell Activation through Intranasal Immunization in Mice.

The effectiveness of vaccines is enhanced by adding adjuvants. Furthermore, the selection of an inoculation route depends on the type of adjuvant used and is important for achieving optimum vaccine efficacy. We investigated the immunological differences between two types of vaccines—spike protein from the Middle East respiratory syndrome virus and inactivated influenza virus vaccine, in combination with a single-stranded RNA adjuvant—administered through various routes (intramuscular, intradermal, and intranasal) to BALB/c mice. Intramuscular immunization with the RNA adjuvant-formulated spike protein elicited the highest humoral immune response, characterized by IgG1 and neutralizing antibody production. Although intranasal immunization did not elicit a humoral response, it showed extensive T-cell activation through large-scale induction of interferon-γ- and interleukin-2-secreting cells, as well as CD4+ T-cell activation in mouse splenocytes. Moreover, only intranasal immunization induced IgA production. When immunized with the inactivated influenza vaccine, administration of the RNA adjuvant via all routes led to protection after viral challenge, regardless of the presence of a vaccine-specific antibody. Therefore, the inoculation route should depend on the type of immune response needed; i.e., the intramuscular route is suitable for eliciting a humoral immune response, whereas the intranasal route is useful for T-cell activation and IgA induction.

Immunization of mice with chimeric antigens displaying selected epitopes confers protection against intestinal colonization and renal damage caused by Shiga toxin-producing Escherichia coli

Shiga toxin-producing Escherichia coli (STEC) cause diarrhea and dysentery, which may progress to hemolytic uremic syndrome (HUS). Vaccination has been proposed as a preventive approach against STEC infection; however, there is no vaccine for humans and those used in animals reduce but do not eliminate the intestinal colonization of STEC. The OmpT, Cah and Hes proteins are widely distributed among clinical STEC strains and are recognized by serum IgG and IgA in patients with HUS. Here, we develop a vaccine formulation based on two chimeric antigens containing epitopes of OmpT, Cah and Hes proteins against STEC strains. Intramuscular and intranasal immunization of mice with these chimeric antigens elicited systemic and local long-lasting humoral responses. However, the class of antibodies generated was dependent on the adjuvant and the route of administration. Moreover, while intramuscular immunization with the combination of the chimeric antigens conferred protection against colonization by STEC O157:H7, the intranasal conferred protection against renal damage caused by STEC O91:H21. This preclinical study supports the potential use of this formulation based on recombinant chimeric proteins as a preventive strategy against STEC infections.

Bioinformatics design and experimental validation of influenza A virus multi-epitopes that induce neutralizing antibodies.

Pandemics caused by influenza A virus (IAV) are responsible for the deaths of millions of humans around the world. One of these pandemics occurred in Mexico in 2009. Despite the impact of IAV on human health, there is no effective vaccine. Gene mutations and translocation of genome segments of different IAV subtypes infecting a single host cell make the development of a universal vaccine difficult. The design of immunogenic peptides using bioinformatics tools could be an interesting strategy to increase the success of vaccines. In this work, we used the predicted amino acid sequences of the neuraminidase (NA) and hemagglutinin (HA) proteins of different IAV subtypes to perform multiple alignments, epitope predictions, molecular dynamics simulations, and experimental validation. Peptide selection was based on the following criteria: promiscuity, protein surface exposure, and the degree of conservation among different medically relevant IAV strains. These peptides were tested using immunological assays to test their ability to induce production of antibodies against IAV. We immunized rabbits and mice and measured the levels of IgG and IgA antibodies in serum samples and nasal washes. Rabbit antibodies against the peptides P11 and P14 (both of which are hybrids of NA and HA) recognized HA from both group 1 (H1, H2, and H5) and group 2 (H3 and H7) IAV and also recognized the purified NA protein from the viral stock (influenza A Puerto Rico/916/34). IgG antibodies from rabbits immunized with P11 and P14 were capable of recognizing viral particles and inhibited virus hemagglutination. Additionally, intranasal immunization of mice with P11 and P14 induced specific IgG and IgA antibodies in serum and nasal mucosa, respectively. Interestingly, the IgG antibodies were found to have neutralizing capability. In conclusion, the peptides designed through in silico studies were validated in experimental assays.Electronic supplementary materialThe online version of this article (10.1007/s00705-020-04537-2) contains supplementary material, which is available to authorized users.