Intramuscular Immunization Research Articles

A single immunization with core–shell structured lipopolyplex mRNA vaccine against rabies induces potent humoral immunity in mice and dogs

ABSTRACT The persistence and clinical consequences of rabies virus (RABV) infection have prompted global efforts to develop a safe and effective vaccines against rabies. mRNA vaccines represent a promising option against emerging and re-emerging infectious diseases, gaining particular interest since the outbreak of COVID-19. Herein, we report the development of a highly efficacious rabies mRNA vaccine composed of sequence-modified mRNA encoding RABV glycoprotein (RABV-G) packaged in core–shell structured lipopolyplex (LPP) nanoparticles, named LPP-mRNA-G. The bilayer structure of LPP improves protection and delivery of RABV-G mRNA and allows gradual release of mRNA molecules as the polymer degrades. The unique core–shell structured nanoparticle of LPP-mRNA-G facilitates vaccine uptake and demonstrates a desirable biodistribution pattern with low liver targeting upon intramuscular immunization. Single administration of low-dose LPP-mRNA-G in mice elicited potent humoral immune response and provided complete protection against intracerebral challenge with lethal RABV. Similarly, single immunization of low-dose LPP-mRNA-G induced high levels of virus-neutralizing antibody titers in dogs. Collectively, our data demonstrate the potential of LPP-mRNA-G as a promising next-generation rabies vaccine used in human and companion animals.

Passive immunization with anti-FimA egg yolk antibodies (IgYs) mitigate Acinetobacter baumannii pneumonia in mice

Acinetobacter baumannii is a formidable pathogen, characterized by high mortality rates and pan-drug-resistant strains. Current commercial antibiotics lack efficacy against drug-resistant variants, necessitating the search for alternative treatments. This study investigates the potential of egg yolk immunoglobulin (IgY) as a cost-effective biomolecule for passive protection against A. baumannii pneumonia. FimA (ABAYE2132), a key virulence factor involved in biofilm development and lung cell adherence, emerges as a promising antigen for triggering protective IgY production. Recombinant FimA was expressed, purified, and used for intramuscular immunization of laying White Leghorn hens. IgY antibodies were subsequently extracted from egg yolks, with their reactivity assessed through indirect ELISA. Neutropenic mice received intranasal administration of IgYs one hour prior to the challenge with a clinical A. baumannii isolate (10 ×LD50). The specific anti-FimA IgYs detected recombinant FimA and provided 100% protection against bacterial infection, while non-specific IgYs prolonged survival for up to 72 h. In contrast, control mice succumbed to infection within 24 h. Analysis of bacterial loads in lungs and spleens after 16 h reveals the following order: control > non-specific IgY > anti-FimA IgY. These findings highlight FimA as a suitable antigen for the development of protective IgYs against A. baumannii.

Antigen-driven T cell-macrophage interactions mediate the interface between innate and adaptive immunity in histidyl-tRNA synthetase-induced myositis.

Previous work in humans has demonstrated that both innate and adaptive immune signaling pathways contribute to the pathogenesis of idiopathic inflammatory myopathy (IIM), a systemic autoimmune disease targeting muscle as well as extra-muscular organs. To better define interactive signaling networks in IIM, we characterized the cellular phenotype and transcriptomic profiles of muscle-infiltrating cells in our established murine model of histidyl-tRNA synthetase (HRS)-induced myositis. Myositis was induced in wild type (WT) and various congenic/mutant strains of C57BL/6 mice through intramuscular immunization with recombinant HRS. Histopathological, immunohistochemical, flow cytometric, and transcriptomic assessments were used to characterize the functional relationship between muscle-infiltrating cell populations in these strains lacking different components of innate and/or adaptive immune signaling. RAG1 KO mice developed markedly reduced muscle inflammation relative to WT mice, demonstrating a key requirement for T cells in driving HRS-induced myositis. While the reduction of mononuclear cell infiltrates in CD4-Cre.MyD88fl/fl conditional knockout mice and OT-II TCR transgenic mice highlighted roles for both innate and TCR-mediated/adaptive immune signaling in T cells, diminished inflammation in Lyz2-Cre.MyD88fl/fl conditional knockout mice underscored the importance of macrophage/myeloid cell populations in supporting T cell infiltration. Single cell RNA sequencing-based clustering of muscle-infiltrating subpopulations and associated pathway analyses showed that perturbations of T cell signaling/function alter the distribution and phenotype of macrophages, fibroblasts, and other non-lymphoid cell populations contributing to HRS-induced myositis. Overall, HRS-induced myositis reflects the complex interplay between multiple cell types that collectively drive a TH1-predominant, pro-inflammatory tissue phenotype requiring antigen-mediated activation of both MyD88- and TCR-dependent T cell signaling pathways.

Broad-spectrum vaccine via combined immunization routes triggers potent immunity to SARS-CoV-2 and its variants.

The development of broad-spectrum SARS-CoV-2 vaccines will reduce the global economic and public health stress from the COVID-19 pandemic. The use of conserved T-cell epitopes in combination with spike antigen that induce humoral and cellular immune responses simultaneously may be a promising strategy to further enhance the broad spectrum of COVID-19 vaccine candidates. Moreover, this research suggests that the combined vaccination strategies have the ability to induce both effective systemic and mucosal immunity, which may represent promising strategies for maximizing the protective efficacy of respiratory virus vaccines.

Immunization with Multiple Virulence Factors Provides Maternal and Neonatal Protection against Group B Streptococcus Serotypes.

Group B streptococcus (GBS) commonly colonizes the vaginal tract and is a leading cause of life-threatening neonatal infections and adverse pregnancy outcomes. No effective vaccine is clinically available. Conserved bacterial virulence factors, including those of GBS, have been employed as vaccine components. We investigated serotype-independent protection against GBS by intranasal immunization with six conserved GBS virulence factors (GBSV6). GBSV6 induced systemic and vaginal antibodies and T cell responses in mice. The immunity reduced mouse mortality and vaginal colonization by various GBS serotypes and protected newborn mice of immunized dams against GBS challenge. Intranasal GBSV6 immunization also provided long-lasting protective immunity and had advantages over intramuscular GBSV6 immunization regarding restricting vaginal GBS colonization. Our findings indicate that intranasal immunization targeting multiple conserved GBS virulence factors induces serotype-independent immunity, which protects against GBS infection systemically and vaginally in dams and prevents newborn death. The study presents valuable strategies for GBS vaccine development.

Boosting of vaginal HSV-2-specific B and T cell responses by intravaginal therapeutic immunization results in diminished recurrent HSV-2 disease.

Boosting herpes simplex virus (HSV)-specific immunity in the genital tissues of HSV-positive individuals to increase control of HSV-2 recurrent disease and virus shedding is an important goal of therapeutic immunization and would impact HSV-2 transmission. Experimental therapeutic HSV-2 vaccines delivered by a parenteral route have resulted in decreased recurrent disease in experimental animals. We used a guinea pig model of HSV-2 infection to test if HSV-specific antibody and cell-mediated responses in the vaginal mucosa would be more effectively increased by intravaginal (Ivag) therapeutic immunization compared to parenteral immunization. Therapeutic immunization with HSV glycoproteins and CpG adjuvant increased glycoprotein-specific IgG titers in vaginal secretions and serum to comparable levels in Ivag- and intramuscular (IM)-immunized animals. However, the mean numbers of HSV glycoprotein-specific antibody secreting cells (ASCs) and IFN-γ SCs were greater in Ivag-immunized animals demonstrating superior boosting of immunity in the vaginal mucosa compared to parenteral immunization. Therapeutic Ivag immunization also resulted in a significant decrease in the cumulative mean lesion days compared to IM immunization. There was no difference in the incidence or magnitude of HSV-2 shedding in either therapeutic immunization group compared to control-treated animals. Collectively, these data demonstrated that Ivag therapeutic immunization was superior compared to parenteral immunization to boost HSV-2 antigen-specific ASC and IFN-γ SC responses in the vagina and control recurrent HSV-2 disease. These results suggest that novel antigen delivery methods providing controlled release of optimized antigen/adjuvant combinations in the vaginal mucosa would be an effective approach for therapeutic HSV vaccines. IMPORTANCE HSV-2 replicates in skin cells before it infects sensory nerve cells where it establishes a lifelong but mostly silent infection. HSV-2 occasionally reactivates, producing new virus which is released back at the skin surface and may be transmitted to new individuals. Some HSV-specific immune cells reside at the skin site of the HSV-2 infection that can quickly activate and clear new virus. Immunizing people already infected with HSV-2 to boost their skin-resident immune cells and rapidly control the new HSV-2 infection is logical, but we do not know the best way to administer the vaccine to achieve this goal. In this study, a therapeutic vaccine given intravaginally resulted in significantly better protection against HSV-2 disease than immunization with the same vaccine by a conventional route. Immunization by the intravaginal route resulted in greater stimulation of vaginal-resident, virus-specific cells that produced antibody and produced immune molecules to rapidly clear virus.

Oral Immunization with rVSV Bivalent Vaccine Elicits Protective Immune Responses, Including ADCC, against Both SARS-CoV-2 and Influenza A Viruses.

COVID-19 and influenza both cause enormous disease burdens, and vaccines are the primary measures for their control. Since these viral diseases are transmitted through the mucosal surface of the respiratory tract, developing an effective and convenient mucosal vaccine should be a high priority. We previously reported a recombinant vesicular stomatitis virus (rVSV)-based bivalent vaccine (v-EM2/SPΔC1Delta) that protects animals from both SARS-CoV-2 and influenza viruses via intramuscular and intranasal immunization. Here, we further investigated the immune response induced by oral immunization with this vaccine and its protective efficacy in mice. The results demonstrated that the oral delivery, like the intranasal route, elicited strong and protective systemic immune responses against SARS-CoV-2 and influenza A virus. This included high levels of neutralizing antibodies (NAbs) against SARS-CoV-2, as well as strong anti-SARS-CoV-2 spike protein (SP) antibody-dependent cellular cytotoxicity (ADCC) and anti-influenza M2 ADCC responses in mice sera. Furthermore, it provided efficient protection against challenge with influenza H1N1 virus in a mouse model, with a 100% survival rate and a significantly low lung viral load of influenza virus. All these findings provide substantial evidence for the effectiveness of oral immunization with the rVSV bivalent vaccine.

Intramuscular Immunization with a Liposomal Multi-Epitope Chimeric Protein Induces Strong Cellular Immune Responses against Visceral Leishmaniasis.

Control of the intracellular parasite Leishmania (L.) requires the activation of strong type 1 cellular immune responses. Towards this goal, in the present study, a multiepitope chimeric protein named LiChimera was encapsulated into cationic liposomes and its protective efficacy against experimental visceral leishmaniasis was investigated. Liposomal LiChimera conferred significant protection against L. infantum as evidenced by the significantly reduced parasite loads in the spleen and liver. Protection detected in Lipo:LiChimera-immunized mice was dependent on the differentiation of long-lasting cellular immune responses and particularly the induction of antigen-specific multifunctional memory CD4+ TH1 and CD8+ T cells that persisted during infection, as evidenced by the persistent high production of IFN-γ and IL-2 and proliferation activity. Notably, protected mice were also characterized by significantly low numbers of non-regulatory CD4+ T cells able to co-produce IFN-γ and IL-10, an important population for disease establishment, as compared to non-immunized control group. Collectively, these results demonstrate that cationic liposomes containing LiChimera can be considered an effective candidate vaccine against visceral leishmaniasis.

ΔA146Ply-HA stem protein immunization protects mice against influenza A virus infection and co-infection with Streptococcus pneumoniae

Influenza virus (IV) is a common pathogen affecting the upper respiratory tract, that causes various diseases. Secondary bacterial pneumonia is a common complication and a major cause of death in influenza patients. Streptococcus pneumoniae (S. pneumoniae) is the predominant co-infected bacteria in the pandemic, which colonizes healthy people but can cause diseases in immunocompromised individuals. Vaccination is a crucial strategy for avoiding infection, however, no universal influenza vaccine (UIV) that is resistant to multiple influenza viruses is available. Despite its limited immunogenicity, the hemagglutinin (HA) stem is a candidate peptide for UIV. ΔA146Ply (pneumolysin with a single deletion of A146) not only retains the Toll-like receptor 4 agonist effect but also is a potential vaccine adjuvant and a candidate protein for the S. pneumoniae vaccine. We constructed the fusion protein ΔA146Ply-HA stem and studied its immunoprotective effect in mice infection models. The results showed that intramuscular immunization of ΔA146Ply-HA stem without adjuvant could induce specific antibodies against HA stem and specific CD4+ T and CD8+ T cellular immunity in BALB/c and C57BL/6 mice, which could improve the survival rate of mice infected with IAV and co-infected with S. pneumoniae, but the protective effect on BALB/c mice was better than that on C57BL/6 mice. ΔA146Ply-HA stem serum antibody could protect BALB/c and C57BL/6 mice from IAV, and recognized HA polypeptides of H3N2, H5N1, H7N9, and H9N2 viruses. Moreover, ΔA146Ply-HA stem intramuscular immunization had a high safety profile with no obvious toxic side effects. The results indicated that coupling ΔA146Ply with influenza protein as a vaccine was a safe and effective strategy against the IV and secondary S. pneumoniae infection.

Comparative safety evaluation of intranasal and intramuscular immunisation with Ad26 and Ad5-vectored vaccines to prevent coronavirus infection

Scientific relevance. Intranasal vaccination may dramatically change current approaches to mass immunisation against various infections, shifting the focus from inducing systemic humoral and cellular immune responses to developing mucosal immunity and protective barriers on the mucous membranes, which are entry points for pathogens. Therefore, the safety of switching from intramuscular to intranasal vaccination should be evaluated.Aim. This study aimed to compare the safety of intranasal and intramuscular vaccination using the intermediate results of the phase III VCI-COV-III clinical trial in healthy volunteers.Materials and methods. The evaluation of the safety profile was based on the adverse events and adverse drug reactions (ADRs) documented by investigators in the interim report on the randomised, double-blind clinical trial of the Salnavac intranasal vaccine (InV) and the Gam-COVID-Vac intramuscular vaccine (ImV) against coronavirus infection. The report covered 42 days of observation in 137 healthy volunteers.Results. ADRs were reported for 17/68 (25%) and 30/69 (43.5%) volunteers in the InV group and the ImV group, respectively (p=0.036). The ADRs reported for the InV group were systemic and local, short-term, mostly mild, and generally consistent in incidence and nature with the ADRs typically observed with other nasal products.Conclusions. According to the preliminary data obtained in the clinical trial, the InV demonstrates a high level of safety. Its safety profile is comparable with those of other intranasal and intramuscular vaccines for the prevention of coronavirus infection.

Investigating the Potential of Cuboidal Nanometals as Protein Subunit Vaccine Carriers In Vivo

AbstractMetal nanoparticles (NPs) are suggested as a vaccine delivery platform. At present, there is limited description of cuboidal Ag nanocubes (AgNCs; nonporous) and Au nanocages (AuNCs; porous) as a protein carrier for vaccination. Here, the intrinsic protein binding ability of AgNC and AuNC is first investigated, using ovalbumin (OVA) as a model antigen, to determine its suitability as a vaccine carrier. Next, the effect of AgNCs and AuNCs on bone‐marrow‐derived dendritic cells (BMDCs) is assessed in vitro. Finally, in vivo humoral and cellular immune responses of AgNC–OVA and AuNC–OVA following intramuscular immunization and their prophylactic effects in B16F10‐OVA mice tumor model are investigated. In terms of OVA loading efficiency, AgNCs are superior to AuNCs. Both nanomaterials are found not to induce BMDC maturation at subtoxic doses. After administration of nanovaccines, serum immunoglobulin G (IgG) responses are comparable between groups. However, there are slight alterations in relative frequencies of lymphocyte subpopulations, with AgNC–OVA‐immunized mice exhibiting lower memory T cells and reduced B cell and T follicular helper cell populations in spleen. Overall, AgNC–OVA and AuNC–OVA immunizations do not alter tumor growth. This study characterizes the intrinsic immunomodulatory properties of AgNCs and AuNCs, as protein subunit vaccine carriers.

Breakthrough infection evokes the nasopharyngeal innate immune responses established by SARS-CoV-2-inactivated vaccine.

Nasopharyngeal immune responses are vital for defense against SARS-CoV-2 infection. Although vaccination via muscle immunization has shown a high efficacy in reducing severity and death in COVID-19 infection, breakthrough infection frequently happens because of mutant variants and incompletely established mucosal immunity, especially in the upper respiratory tract. Here, we performed a single-cell RNA and T-cell receptor repertoire sequencing and delineated a high-resolution transcriptome landscape of nasopharyngeal mucosal immune and epithelial cells in vaccinated persons with breakthrough infection and non-vaccinated persons with natural infection as control. The epithelial cells showed anti-virus gene expression diversity and potentially recruited innate immune cells into the nasopharyngeal mucous of vaccinated patients. Upon infection, they released significant pro-inflammatory cytokines and chemokines by macrophages and monocytes and expressed antigen-presenting relevant genes by dendritic cells. Such immune responses of nasopharyngeal innate immune cells would facilitate the strengthened expression of cytotoxic genes in virus-specific T-cell or B-cell differentiation into antibody-secreting cells at the early stage of breakthrough infection through cell interaction between innate and adaptive immune cells. Notably, these alterations of nasopharyngeal immune cells in breakthrough infection depended on the activated Nuclear factor-κB (NF-κB) and NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) signaling rather than type I interferon responses due to the general reduction in interferon-stimulated gene expression. Our findings suggest that vaccination potentially strengthens innate immune barriers and virus-specific memory immune cell responses, which could be quickly activated to defend against variant breakthrough infection and maintain nasopharyngeal epithelial cell integrity. Thus, this study highlights the necessity of a boost via nasal mucous after intramuscular immunization.

Evaluation of Four Adjuvant Combinations, IVAX-1, IVAX-2, CpG-1826+Montanide ISA 720 VG and CpG-1018+Montanide ISA 720 VG, for Safety and for Their Ability to Elicit Protective Immune Responses in Mice against a Respiratory Challenge with Chlamydia muridarum.

There is an urgent need to produce a vaccine for Chlamydia trachomatis infections. Here, using the Chlamydia muridarum major outer membrane protein (MOMP) as an antigen, four adjuvant combinations IVAX-1 (MPLA+CpG-1018+AddaVax), IVAX-2 (MPLA+CpG-1018+AS03), CpG-1826+Montanide ISA 720 VG (CpG-1826+Mont) and CpG-1018+Montanide ISA 720 VG (CpG-1018+Mont), were tested for their local reactogenicity and ability to elicit protection in BALB/c mice against a respiratory challenge with C. muridarum. Immunization with IVAX-1 or IVAX-2 induced no significant local reactogenicity following intramuscular immunization. In contrast, vaccines containing Montanide resulted in the formation of a local granuloma. Based on the IgG2a/IgG1 ratio in serum, the four adjuvant combinations elicited Th1-biased responses. IVAX-1 induced the highest in vitro neutralization titers while CpG-1018+Mont stimulated the lowest. As determined by the levels of IFN-γ produced by T-cells, the most robust cellular immune responses were elicited in mice immunized with CpG-1018+Mont, while the weakest responses were mounted by mice receiving IVAX-1. Following the respiratory challenge, mice immunized with CpG-1018+Mont lost the least amount of body weight and had the lowest number of C. muridarum inclusion-forming units (IFUs) in the lungs, while those receiving IVAX-2 had lost the most weight and had the highest number of IFUs in their lungs. Animals vaccinated with CpG-1826+Mont had the lightest lungs while those immunized using IVAX-2 had the heaviest. To conclude, due to their safety and adjuvanticity, IVAX formulations should be considered for inclusion in human vaccines against Chlamydia.

Self-assembled ferritin nanoparticles displaying PcrV and OprI as an adjuvant-free Pseudomonas aeruginosa vaccine.

Serious infections of Pseudomonas aeruginosa (PA) in hospitals and the emergence and increase of multidrug resistance have raised an urgent need for effective vaccines. However, no vaccine has been approved to date. One possible reason for this is the limited immune response due to the lack of an efficient delivery system. Self-assembled ferritin nanoparticles are good carriers of heterogeneous antigens, which enhance the activation of immunological responses. In this study, two well-studied antigen candidates, PcrV and OprI, were selected and connected to the ferritin nanoparticle by the Spytag/SpyCatcher system to generate the nanovaccine rePO-FN. Compared to recombinant PcrV-OprI formulated with aluminum adjuvants, intramuscular immunization with adjuvant-free rePO-FN induced quick and efficient immunity and conferred protection against PA pneumonia in mice. In addition, intranasal immunization with adjuvant-free rePO-FN enhanced protective mucosal immunity. Moreover, rePO-FN exhibited good biocompatibility and safety. Our results suggest that rePO-FN is a promising vaccine candidate, as well as, provide additional evidence for the success of ferritin-based nanovaccines.

Dual pattern recognition receptor ligands CL401, CL413, and CL429 as adjuvants for inactivated chikungunya virus

Chikungunya virus (CHIKV) is responsible for incapacitating joint pains and is a significant health hazard in many countries. Though a definite need for a CHIKV vaccine is felt, long disappearance of CHIKV from circulation in humans has been a concern for vaccine development. Use of two separate pattern recognition receptor ligands has been shown to enhance immune response to the administered antigen. In addition, intradermal delivery of vaccine tends to mimic the natural mode of CHIKV infection. Therefore, in this study, we explored whether intradermal and intramuscular immunization with inactivated CHIKV (I–CHIKV) supplemented with dual pattern-recognition receptor ligands, CL401, CL413, and CL429, is an effective approach to enhancing antibody response to CHIKV. Our in vivo data show that I–CHIKV supplemented with these chimeric PRR ligands induces enhanced neutralizing antibody response after intradermal delivery, but is less efficient after intramuscular immunization. These results suggest that intradermal delivery of I–CHIKV with chimeric adjuvants is a possible way to elicited a better antibody response.

Recombinant BoHV-5 glycoprotein (rgD5) elicits long-lasting protective immunity in cattle

BoHV-5 is a worldwide distributed pathogen usually associated with a lethal neurological disease in dairy and beef cattle resulting in important economic losses due to the cattle industry. Using recombinant gD5, we evaluated the long-duration humoral immunity of the recombinant vaccines in a cattle model. Here we report that two doses of intramuscular immunization, particularly with the rgD5ISA vaccine, induce long-lasting antibody responses. Recombinant gD5 antigen elicited tightly mRNA transcription of the Bcl6 and the chemokine receptor CXCR5 which mediate memory B cells and long-lived plasma cells in germinal centers. In addition, using an in-house indirect ELISA we observed higher and earlier responses of rgD5-specific IgG antibody and the upregulation of mRNA transcription of IL2, IL4, IL10, IL15, and IFN-γ in rgD5 vaccinated cattle, indicating a mixed immune response. We further show that rgD5 immunization protects against both BoHV −1 and −5. Our findings indicate that the rgD5-based vaccine represents an effective vaccine strategy to induce an efficient control of herpesviruses.

Investigating the potential of silver nanocubes and gold nanocages as protein subunit vaccine carriers

Abstract Metal nanoparticles have been exploited as vaccine delivery platforms for various diseases. In particular, Ag and Au NPs of different size and shape have been shown to promote delivery of antigens and act as efficient adjuvants. Yet, there is limited description of cuboidal Ag nanocubes (AgNCs) and Au nanocages (AuNCs) as antigen carriers for vaccination. Here, we first investigated the intrinsic protein binding ability of AgNCs and AuNCs, using ovalbumin (OVA) as a model. Next, the effect of AgNCs and AuNCs on bone marrow-derived dendritic cells (BMDCs) in terms of cytotoxicity and maturation was assessed. Finally, we investigated humoral and cellular immune responses of AgNCs-OVA and AuNCs-OVA following intramuscular immunization and their prophylactic effects in B16F10-OVA mice tumor model. In terms of OVA loading efficiency, AgNCs were more potent than AuNCs, despite their non-porous form. Both cubic nanomaterials were found not to induce BMDCs maturation in vitro or DCs maturation in spleen. Following the administration of nanovaccine doses, serum IgG responses were comparable between groups. However, there were significant alterations in relative frequencies of lymphocyte sub-populations. Overall, AgNCs-OVA and AuNCs-OVA immunization via intramuscular route did not alter tumour growth. This study has demonstrated the intrinsic immunomodulatory properties of AgNCs and AuNCs, preceding their potential use as nanocarriers. Supported by grants from British Council (Newton Fund, 337313), Wellcome Trust (WT103913), and TUBITAK.

Oral and intranasal Ad5 influenza vaccination induces hemagglutinin-specific and influenza neutralizing antibodies in serum and milk of pregnant and lactating ferrets

Abstract Pregnant women and young children are at increased risk for severe influenza infection. While there are intramuscular (IM) inactivated influenza vaccines available to pregnant individuals, IM immunization may not be an ideal route to boost neutralizing antibodies in breastmilk (BM). Therefore, innovative vaccine strategies are needed to increase BM antibodies that promote neonatal protection against influenza. We hypothesize that maternal mucosal immunization stimulates a protective influenza-specific antibody response, by generating antibodies in both blood and BM. To test this, we immunized pregnant and lactating ferrets at two weeks prepartum and two weeks postpartum with an H1N1 hemagglutinin (HA) adenovirus type 5 (Ad5) vaccine via oral or intranasal routes. We detected HA-specific IgG antibodies in dam serum and HA-specific IgG and IgA antibodies in secreted milk after oral and intranasal immunization by ELISA and chemiluminescence assays. We demonstrated that serum and BM from oral and intranasally immunized dams neutralized influenza virus in vitro. Additionally, ferret kits born to oral and intanasally immunized dams had HA-specific IgG antibodies in circulation at 5–6 weeks of life. These preclinical data led to the announcement by Vaxart, Inc. that they will test their oral tablet vaccine in human lactating mothers to determine if vaccination induces BM antibodies. Future work includes using our ferret model to determine the relationship between BM antibody quantity and function and neonatal protection against influenza. An easy-to-deliver mucosal vaccine that boosts systemic and mucosal protective antibodies in the maternal-neonatal dyad is an ideal strategy for protection of these vulnerable populations. Bill and Melinda Gates Foundation grant INV-22595D

Adenoviral vector vaccines are effective mucosal booster modalities to elevate systemic and establish mucosal immunity against SARS-CoV-2

Abstract Licensed SARS-CoV-2 vaccines induce robust systemic immune responses, but mucosal immunity in the respiratory tract in form of secretory immunoglobulin A (IgA) or tissue-resident memory T cells (TRM) is not established. Inducing such front-line immunity by mucosal boosting might reduce the individual risk of breakthrough infections and thereby likely mitigate infection dynamics on population level as well. The following project evaluates preclinical and licensed adenoviral vector (Ad) platforms as mucosal SARS-CoV-2 boosters. We show that nasal instillation of Ad5- and Ad19a-Spike following a systemic DNA or mRNA priming results in strong systemic and mucosal immunity in mice. In contrast to two intramuscular injections with a licensed mRNA vaccine, the mucosal boost with Ad vectors induced CD103 +CD69 +tissue-resident memory CD8 +T cells and high levels of Spike-specific IgA in the respiratory tract as well as an enhanced mucosal neutralization of variants of concern. Moreover, the protective efficacy of the mRNA prime/intranasal Ad5 boost strategy was not inferior to two intramuscular mRNA immunizations. In a more translational approach, we employed Janssen’s Ad26.COV2.S as a mucosal booster delivered by a medical mucosal atomization device after a systemic mRNA prime of mice. Concerning systemic responses, the mucosal boost was similarly immunogenic compared to an intramuscular boost with the same vaccine. However, only a mucosal delivery provoked TRM and IgA in the respiratory tract as well as a robust mucosal neutralization of Omicron variants. In conclusion, a mucosal booster immunization is a promising approach to establish mucosal immunity in addition to systemic responses in an mRNA vaccine-primed population.

ISCOMs/MPLA adjuvanted SDAD protein nanoparticles induce improved immune responses and cross-protection in mice

Abstract The development of a universal influenza vaccine to protect against influenza variants will be the priority goal in combating influenza virus infection. In this study, we exploited a novel type of protein nanoparticles consisting of influenza NP as the core and NA1-M2e or NA2-M2e fusion proteins as the coating antigens by using an SDAD heterobifunctional crosslinker. Intramuscular immunization of the NP/NA-M2e (core/shell) nanoparticles induced increased humoral and cellular immune responses. ISCOMs/MPLA as adjuvants further improved the nanoparticle immunogenicity and elicited stronger immune reactions conferring protection against different influenza viral challenges. The nanoparticles were delivered through intranasal route to determine the application as a mucosal vaccine. While the nanoparticles alone barely induced immune response after intranasal immunization, ISCOMs/MPLA adjuvanted nanoparticles induced strengthened antigens-specific antibodies responses, cellular responses and higher levels of mucosal IgA. Meanwhile, increased lung Trm, Brm , and alveolar macrophages, were observed from ISCOMs/MPLA adjuvanted nanoparticle group. Only the mice intranasally vaccinated with ISCOMs/MPLA adjuvanted nanoparticles fully survived during the infections, and better protection efficacy was observed when compared with other adjuvants. Our results emphasized the importance of supplementing of appropriate adjuvants to improve the immunogenicity and mucosal immune responses of vaccines in mucosal immunization. In conclusion, the ISCOMs/MPLA adjuvant combination could significantly improve the immune responses and protective efficiency of protein nanoparticles in different immunization routes.