Vaccine Formulations Research Articles

The N and C-terminal deleted variant of the dengue virus NS1 protein is a potential candidate for dengue vaccine development

NS1 is an elusive dengue protein, involved in viral replication, assembly, pathogenesis, and immune evasion. Its levels in blood plasm are positively related to disease severity like thrombocytopenia, hemorrhage, and vascular leakage. Despite its pathogenic roles, NS1 is being used in various vaccine formulations due to its sequence conservancy, ability to produce protective antibodies and low risk for inducing antibody-dependent enhancement. In this study, we have used bioinformatics tools and reported literature to develop an NS1 variant (dNS1). Molecular docking studies were performed to evaluate the receptor-binding ability of the NS1 and dNS1 with TLR4. NS1 and dNS1 (153 to 312 amino acid region) genes were cloned, expressed and protein was purified followed by refolding. Docking studies showed the binding of NS1 and dNS1 with the TLR4 receptor which suggests that N and C-terminal sequences of NS1 are not critical for receptor binding. Antibodies against NS1 and dNS1 were raised in rabbits and binding affinity of anti-dNS1 anti-NS1 sera was evaluated against both NS1 and dNS1. Similar results were observed through western blotting which highlight that N and C-terminal deletion of NS1 does not compromise the immunogenic potential of dNS1 hence, supports its use in future vaccine formulations as a substitute for NS1.

Virus-like particles displaying the mature C-terminal domain of filamentous hemagglutinin are immunogenic and protective against Bordetella pertussis respiratory infection in mice.

Bordetella pertussis, the bacterium responsible for whooping cough, remains a significant public health challenge despite the existing licensed pertussis vaccines. Current acellular pertussis vaccines, though having favorable reactogenicity and efficacy profiles, involve complex and costly production processes. In addition, acellular vaccines have functional challenges such as short-lasting duration of immunity and limited antigen coverage. Filamentous hemagglutinin (FHA) is an adhesin of B. pertussis that is included in all multivalent pertussis vaccine formulations. Antibodies to FHA have been shown to prevent bacterial attachment to respiratory epithelial cells, and T cell responses to FHA facilitate cell-mediated immunity. In this study, FHA's mature C-terminal domain (MCD) was evaluated as a novel vaccine antigen. MCD was conjugated to virus-like particles via SpyTag-SpyCatcher technology. Prime-boost vaccine studies were performed in mice to characterize immunogenicity and protection against the intranasal B. pertussis challenge. MCD-SpyVLP was more immunogenic than SpyTag-MCD antigen alone, and in Tohama I strain challenge studies, improved protection against challenge was observed in the lungs at day 3 and in the trachea and nasal wash at day 7 post-challenge. Furthermore, a B. pertussis strain encoding genetically inactivated pertussis toxin was used to evaluate MCD-SpyVLP vaccine immunity. Mice vaccinated with MCD-SpyVLP had significantly lower respiratory bacterial burden at both days 3 and 7 post-challenge compared to mock-vaccinated animals. Overall, these data support the use of SpyTag-SpyCatcher VLPs as a platform for use in vaccine development against B. pertussis and other pathogens.

Epidemiology and Impact of Anti-Pneumococcal Vaccination and COVID-19 on Resistance of Streptococcus pneumoniae Causing Invasive Disease in Piedmont, Italy.

The international surveillance of antimicrobial resistance (AMR) reports S. pneumoniae as one of leading causes of death associated with AMR. Against invasive disease, several vaccinations are available and a reduction in AMR in S. pneumoniae has been observed. Here, we evaluated the impact of anti-pneumococcal vaccination policy and the SARS-CoV2 outbreak on AMR in S. pneumoniae causing invasive disease. We collected all strains of S. pneumoniae causing invasive disease from 2008 in the Piedmont region (Italy). Each strain was typed in order to identify the serogroup and data about AMR were collected. The population under surveillance was classified as infants, children, adults, and the old population. We collected n = 2076 S. pneumoniae strains, with 21.9% and 40.3% being resistant to penicillin G and erythromycin, respectively. We reported an increased risk of infection with penicillin-resistant S. pneumoniae among all populations and evaluated whether the infection was caused by a serotype included in the vaccine formulation. A similar increase was observed after the SARS-CoV2 outbreak. In the Piedmont region, subsequently to the introduction of anti-pneumococcal vaccination, a significant increase in the risk of penicillin G-resistant invasive pneumococcal disease among infants and old population was reported. No significant impact was found for the SARS-CoV2 outbreak.

Impact of vaccination with the Anaplasma phagocytophilum MSP4 chimeric antigen on gene expression in the rabbit host

There are currently no vaccines available to prevent and control of Anaplasma phagocytophilum, an intracellular bacterial pathogen transmitted by ticks that occurs in many regions of the world and causes disease in a wide range of domestic and wild hosts, including humans. Vaccines induce long-lasting immunity and could prevent or reduce transmission of this pathogen.Understanding how vaccines induce a protective response can be difficult due to the complexity of the immune system, which operates at many levels throughout the organism. New perspectives in vaccinology, based on systems biology approaches, integrate many scientific disciplines to fully understand the biological responses to vaccination, where a transcriptomic approach could reveal relevant information of the host immune system, allowing profiling for rational design of vaccine formulations, administration, and potential protection.In the present study we report the gene expression profiles by RNA-seq followed by functional analysis using whole blood samples from rabbits immunized with a recombinant chimeric protein containing peptides from the MSP4 protein of A. phagocytophilum, which showed satisfactory results in terms of potential protection. Transcriptomic analysis revealed differential expression of 720 genes, with 346 genes upregulated and 374 genes downregulated. Overrepresentation of biological and metabolic pathways correlated with immune response, protein signaling, cytoskeleton organization and protein synthesis were found. These changes in gene expression could provide a complete and unique picture of the biological response to the epitope candidate vaccine against A. phagocytophilum in the host.

Heterologous mRNA/MVA delivering trimeric-RBD as effective vaccination regimen against SARS-CoV-2: COVARNA Consortium

ABSTRACT Despite the high efficiency of current SARS-CoV-2 mRNA vaccines in reducing COVID-19 morbidity and mortality, waning immunity and the emergence of resistant variants underscore the need for novel vaccination strategies. This study explores a heterologous mRNA/Modified Vaccinia virus Ankara (MVA) prime/boost regimen employing a trimeric form of the receptor binding domain (RBD) of the SARS-CoV-2 spike (S) protein compared to a homologous MVA/MVA regimen. In C57BL/6 mice, the RBD was delivered during priming via an mRNA vector encapsulated in nanoemulsions (NE) or lipid nanoparticles (LNP), followed by a booster with a replication-deficient MVA-based recombinant virus (MVA-RBD). This heterologous mRNA/MVA regimen elicited strong anti-RBD binding and neutralizing antibodies (BAbs and NAbs) against both the ancestral SARS-CoV-2 strain and different variants of concern (VoCs). Additionally, this protocol induced robust and polyfunctional RBD-specific CD4 and CD8 T cell responses, particularly in animals primed with mLNP-RBD. In K18-hACE2 transgenic mice, the LNP-RBD/MVA combination provided complete protection from morbidity and mortality following a live SARS-CoV-2 challenge compared with the partial protection observed with mNE-RBD/MVA or MVA/MVA regimens. Although the mNE-RBD/MVA regimen only protects half of the animals, it was able to induce antibodies with Fc-mediated effector functions besides NAbs. Moreover, viral replication and viral load in the respiratory tract were markedly reduced and decreased pro-inflammatory cytokine levels were observed. These results support the efficacy of heterologous mRNA/MVA vaccine combinations over homologous MVA/MVA regimen, using alternative nanocarriers that circumvent intellectual property restrictions of current mRNA vaccine formulations.

Immunomodulatory activity of polycaprolactone nanoparticles with calcium phosphate salts against Leishmania infantum infection

Objective: To prepare and characterize polycaprolactone (PCL) nanoparticles loaded with sonicator fragmented (SLA) and freeze- thaw Leishmania antigens (FTLA) and to investigate the in vitro immunogenicity of antigen-encapsulated nanoparticles with calcium phosphate adjuvant. Methods: The water/oil/water binary emulsion solvent evaporation method was used to synthesize antigen-loaded PCL nanoparticles. Particles were characterized by scanning electron microscopy and zeta potential measurements. Their cytotoxicity in J774 macrophages in vitro was determined by MTT analysis. In addition, the amount of nitric oxide and the level of cytokines produced by macrophages were determined by Griess reaction and ELISA method, respectively. The protective effect of the developed formulations was evaluated by determining the infection index percentage in macrophages infected with Leishmania infantum. Results: Compared to the control group, SLA PCL and FTLA PCL nanoparticles with calcium phosphate adjuvant induced a 6- and 7-fold increase in nitric oxide, respectively. Additionally, the vaccine formulations promoted the production of IFN-γ and IL-12. SLA PCL and FTLA PCL nanoparticles combined with calcium phosphate adjuvant caused an approximately 13- and 11-fold reduction in infection index, respectively, compared to the control group. Conclusions: The encapsulation of antigens obtained by both sonication and freeze-thawing into PCL nanoparticles and the formulations with calcium phosphate adjuvant show strong in vitro immune stimulating properties. Therefore, PCL-based antigen delivery systems and calcium phosphate adjuvant are recommended as a potential vaccine candidate against leishmaniasis.

Lysosomal Activation Mediated by Endocytosis in J774 Cell Culture Treated with N-Trimethyl Chitosan Nanoparticles.

Safety and effectiveness are the cornerstone objectives of nanomedicine in developing nanotherapies. It is crucial to understand the biological interactions between nanoparticles and immune cells. This study focuses on the manufacture by the microfluidic technique of N-trimethyl chitosan/protein nanocarriers and their interaction with J774 cells to elucidate the cellular processes involved in absorption and their impact on the immune system, mainly through endocytosis, activation of lysosomes and intracellular degradation. TEM of the manufactured nanoparticles showed spherical morphology with an average diameter ranging from 36 ± 16 nm to 179 ± 92 nm, depending on the concentration of the cargo protein (0, 12, 55 μg/mL). FTIR showed the crosslinking between N-trimethyl chitosan and the sodium tripolyphosphate and the α-helix binding loss of BSA. TGA revealed an increase in the thermal stability of N-trimethyl chitosan/protein nanoparticles compared with the powder. The encapsulation of the cargo protein used was demonstrated using XPS. Their potential to improve cell permeability and use as nanocarriers in future vaccine formulations was demonstrated. The toxicity of the nanoparticles in HaCaT and J774 cells was studied, as well as the importance of evaluating the differentiation status of J774 cells. Thus, possible endocytosis pathways and their impact on the immune response were discussed. This allowed us to conclude that N-trimethyl chitosan nanoparticles show potential as carriers for the immune system. Still, more studies are required to understand their effectiveness and possible use in therapies.

Evaluation of the adjuvant effect of imiquimod and CpG ODN 1826 in chimeric DNA vaccine against Japanese encephalitis

Vaccines represent a significant milestone in the history of human medical science and serve as the primary means for controlling infectious diseases. In recent years, the geographical distribution of Japanese encephalitis viruses (JEV) of various genotypes has become increasingly complex, which provides a rationale for the development of safer and more effective vaccines. The advent of subunit and nucleic acid vaccines, especially propelled by advancements in genetic engineering since the 1980s, has accelerated the application of novel adjuvants. These novel vaccine adjuvants have diversified into toll-like receptor (TLR) agonists, complex adjuvants, nanoparticles and so on. However, the efficacy of adjuvant combinations can vary depending on the host system, disease model, or vaccine formulation, sometimes resulting in competitive or counteractive effects. In our previous study, we constructed a pJME-LC3 chimeric DNA vaccine aimed at inducing an immune response through autophagy induction. Building on this, we investigated the impact of the TLR7/8 agonist imiquimod (IMQ) and the TLR9 agonist CpG ODN 1826 as adjuvants on the immunogenicity of the Japanese encephalitis chimeric DNA vaccine. Our findings indicate that the combination of the pJME-LC3 vaccine with IMQ and CpG ODN 1826 adjuvants enhanced the innate immune response, promoting the maturation and activation of antigen-presenting cells in the early immune response. Furthermore, it played a regulatory and optimizing role in subsequent antigen-specific immune responses, resulting in effective cellular and humoral immunity and providing prolonged immune protection. The synergistic effect of IMQ and CpG ODN 1826 as adjuvants offers a novel approach for the development of Japanese encephalitis nucleic acid vaccines.

Development of multidose thermotolerant formulations of a vector-based Covid-19 vaccine candidate, NDV-HXP-S in different product formats: Stability and preservative efficacy study

Current lead coronavirus vaccines require continuous cold or ultra-cold storage from the manufacturing site to the field to maintain protective efficacy. Since cold chain capacity is limited and complex, logistics planning is crucial to limit vaccine wastage.[1] The restrictive storage concerns also make it difficult to share vaccines between public health departments and neighboring states, leading to increased vaccine wastage.[2] A Newcastle Disease Virus (NDV) vector-based severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) vaccine candidate, NDV-HXP-S, offers a cost-effective alternative which aims to improve global access to SARS CoV-2 vaccines.[3] The NDV-HXP-S vaccine candidate can be mass-produced in chicken eggs and has demonstrated efficacy in preclinical studies, as well as acceptable safety andpotent immunogenicity in clinical studies.[3,4–10] To further advance the NDV-HXP-S vaccine candidate, this manuscript describes work focused on the development of multidose thermotolerant vaccine formulations (i.e., those which would not require continuous extendedrefrigeration), making it convenient to use and store, and simplifying transport and distribution logistics, especially in outbreak settings. Liquid and lyophilized formulations for parenteral administration were rigorously screened for the vaccine formulation’s ability to maintain S-antigen stability after exposure to temperaturestress at 40 °C, 25 °C, and 2 °C to 8 °C storage for six months. Preservative efficacy was evaluated to enable a multidose liquid vaccine format as well as endotoxin testing in lyophilized formulations. Lead liquid vaccine formations were identified that were able to maintain S-antigen content at 2 °C to 8 °C and 25 °C storage for the entire six-month study. Lead lyophilized vaccine formulations were identified which were able to maintain S-antigen content for six months at 2 °C to 8 °C, 25 °C, and 40 °C. Both the liquid and lyophilized formulations identified are improved thermotolerant SARS-CoV-2 vaccine formulations.

Investigation of cross-opsonic effect leads to the discovery of PPIase-domain containing protein vaccine candidate to prevent infections by Gram-positive ESKAPE pathogens

BackgroundEnterococcus faecium and Staphylococcus aureus are the Gram-positive pathogens of the ESKAPE group, known to represent a great threat to human health due to their high virulence and multiple resistances to antibiotics. Combined, enterococci and S. aureus account for 26% of healthcare-associated infections and are the most common organisms responsible for blood stream infections. We previously showed that the peptidyl-prolyl cis/trans isomerase (PPIase) PpiC of E. faecium elicits the production of specific, opsonic, and protective antibodies that are effective against several strains of E. faecium and E. faecalis. Due to the ubiquitous characteristics of PPIases and their essential function within Gram-positive cells, we hypothesized a potential cross-reactive effect of anti-PpiC antibodies.ResultsOpsonophagocytic assays combined with bioinformatics led to the identification of the foldase protein PrsA as a new potential vaccine antigen in S. aureus. We show that PrsA is a stable dimeric protein able to elicit opsonic antibodies against the S. aureus strain MW2, as well as cross-binding and cross-opsonic in several S. aureus, E. faecium and E. faecalis strains.ConclusionsGiven the multiple antibiotic resistances S. aureus and enterococci present, finding preventive strategies is essential to fight those two nosocomial pathogens. The study shows the potential of PrsA as an antigen to use in vaccine formulation against the two dangerous Gram-positive ESKAPE bacteria. Our findings support the idea that PPIases should be further investigated as vaccine targets in the frame of pan-vaccinomics strategy.

Immunogenicity of an Inactivated Senecavirus A Vaccine with a Contemporary Brazilian Strain in Mice.

Senecavirus A (SVA) is a picornavirus that is endemic in swine, causing a vesicular disease clinically indistinguishable from other vesicular diseases, like foot-and-mouth disease. The widespread viral circulation, constant evolution, and economic losses caused to the swine industry emphasize the need for measures to control the agent. In this study, we evaluated the immunogenicity of a whole-virus-inactivated vaccine using a representative contemporary Brazilian SVA strain in Balb/ByJ mice. The animals were vaccinated with two doses by an intramuscular route. The humoral response induced by the vaccination was evaluated by an in-house ELISA assay for IgG detection. The cellular response was assessed by flow cytometry after in vitro SVA stimulation in splenocyte cultures from vaccinated and non-vaccinated groups. Protection against SVA was assessed in the experimental groups following an oral challenge with the homologous virus. The vaccination induced high levels of IgG antibodies and the proliferation of CD45R/B220+sIgM+, CD3e+CD69+, and CD3e+CD4+CD44+CD62L- cells. These results indicate the immunogenicity and safety of the vaccine formulation in a murine model and the induction of humoral and cellular response against SVA.

Formulation Development of a COVID-19 Recombinant Spike Protein-Based Vaccine.

The purpose of this study was to develop a formulation for a recombinant prefusion spike protein vaccine against SARS-CoV-2. It was found that the spike protein was susceptible to aggregation due to mechanical stress. Therefore, formulation studies were initiated focused on screening pharmaceutical excipients capable of preventing this. The screening of a panel of potential stabilizing conditions found that Tween 20 could inhibit mechanically induced aggregation. A concentration-dependent study indicated that a higher concentration of Tween 20 (0.2% v/v) was required to prevent conformational changes in the trimer. The conformational changes induced by mechanical stress were characterized by size exclusion chromatography (SEC) and hydrogen-deuterium exchange mass spectrometry (HDX-MS), indicating the formation of an extended trimeric conformation that was also unable to bind to antibodies directed to the S2 domain. Long-term stability modeling, using advanced kinetic analysis, indicated that the formulation containing 0.2% (v/v) Tween 20 at a neutral pH was predicted to be stable for at least two years at 2 °C to 8 °C. Additional stabilizer screening conducted by thermal shift assay indicated that sucrose and glycerol were able to significantly increase the spike protein melting temperature (Tm) and improve the overall thermostability of the spike protein in a short-term stability study. Thus, while 0.2% (v/v) Tween 20 was sufficient to prevent aggregation and to maintain spike protein stability under refrigeration, the addition of sucrose further improved vaccine thermostability. Altogether, our study provides a systematic approach to the formulation of protein-based COVID-19 vaccine and highlights the impact of mechanical stress on the conformation of the spike protein and the significance of surfactants and stabilizers in maintaining the structural and functional integrity of the spike protein.

Chimeric lipoproteins for leptospirosis vaccine: immunogenicity and protective potential

Leptospirosis, a neglected zoonotic disease, is caused by pathogenic spirochetes belonging to the genus Leptospira and has one of the highest morbidity and mortality rates worldwide. Vaccination stands out as one of the most effective preventive measures for susceptible populations. Within the outer membrane of Leptospira spp., we find the LIC12287, LIC11711, and LIC13259 lipoproteins. These are of interest due to their surface location and potential immunogenicity. Thorough examination revealed the conservation of these proteins among pathogenic Leptospira spp.; we mapped the distribution of T- and B-cell epitopes along their sequences and assessed the 3D structures of each protein. This information aided in selecting immunodominant regions for the development of a chimeric protein. Through gene synthesis, we successfully constructed a chimeric protein, which was subsequently expressed, purified, and characterized. Hamsters were immunized with the chimeric lipoprotein, formulated with adjuvants aluminum hydroxide, EMULSIGEN®-D, Sigma Adjuvant System®, and Montanide™ ISA206VG. Another group was vaccinated with an inactivated Escherichia coli bacterin expressing the chimeric protein. Following vaccination, hamsters were challenged with a virulent L. interrogans strain. Our evaluation of the humoral immune response revealed the production of IgG antibodies, detectable 28 days after the second dose, in contrast to pre-immune samples and control groups. This demonstrates the potential of the chimeric protein to elicit a robust humoral immune response; however, no protection against challenge was achieved. While this study provides valuable insights into the subject, further research is warranted to identify protective antigens that could be utilized in the development of a leptospirosis vaccine.Key points• Several T- and B-cell epitopes were identified in all the three proteins.• Four different adjuvants were used in vaccine formulations.• Immunization stimulated significant levels of IgG2/3 in vaccinated animals.

Relationship between Endotoxin Content in Vaccine Preclinical Formulations and Animal Welfare: An Extensive Study on Historical Data to Set an Informed Threshold.

The most widely known pyrogen impurity in vaccines is the Gram-negative bacterial endotoxin lipopolysaccharide (LPS). When administered at toxic doses, endotoxin triggers inflammatory responses, which lead to endotoxic shock. The literature on endotoxic content (EC) for preclinical vaccines' formulations used in animal studies is very poor, and the recommended thresholds are solely based on commercial vaccine limits set for humans and are, therefore, not connected to the actual impact of EC on animal welfare for species used in preclinical research studies. An extensive study to evaluate the presence of a potential relationship between endotoxin content in formulations administered to mice (the most common species used in preclinical research studies) and their welfare was conducted to calculate an EC threshold for formulations of candidate vaccines. Three years of historical data, from more than 500 formulations of different antigen types (i.e., proteins, glycoconjugates, OMV/GMMA) injected into more than 5000 mice, was evaluated with two alternative statistical methodologies, both demonstrating that there is no significant relationship between actual endotoxin levels and mouse welfare. The calculation of thresholds was, therefore, performed by consistency versus formulations that demonstrated no impact on animal welfare.

Toll-like receptor 4 (TLR4) is the major pattern recognition receptor triggering the protective effect of a Candida albicans extracellular vesicle-based vaccine prototype in murine systemic candidiasis.

Systemic candidiasis remains a significant public health concern worldwide, with high mortality rates despite available antifungal drugs. Drug-resistant strains add to the urgency for alternative therapies. In this context, vaccination has reemerged as a prominent immune-based strategy. Extracellular vesicles (EVs), nanosized lipid bilayer particles, carry a diverse array of native fungal antigens, including proteins, nucleic acids, lipids, and glycans. Previous studies from our laboratory demonstrated that Candida albicans EVs triggered the innate immune response, activating bone marrow-derived dendritic cells (BMDCs) and potentially acting as a bridge between innate and adaptive immunity. Vaccination with C. albicans EVs induced the production of specific antibodies, modulated cytokine production, and provided protection in immunosuppressed mice infected with lethal C. albicans inoculum. To elucidate the mechanisms underlying EV-induced immune activation, our study investigated pathogen-associated molecular patterns (PAMPs) and pattern recognition receptors (PRRs) involved in EVs-phagocyte engagement. EVs from wild-type and mutant C. albicans strains with truncated mannoproteins were compared for their ability to stimulate BMDCs. Our findings revealed that EV decoration with O- and N-linked mannans and the presence of β-1,3-glucans and chitin oligomers may modulate the activation of specific PRRs, in particular Toll-like receptor 4 (TLR4) and dectin-1. The protective effect of vaccination with wild-type EVs was found to be dependent on TLR4. These results suggest that fungal EVs can be harnessed in vaccine formulations to selectively activate PRRs in phagocytes, offering potential avenues for combating or preventing candidiasis.IMPORTANCESystemic candidiasis is a serious global health concern with high mortality rates and growing drug resistance. Vaccination offers a promising solution. A unique approach involves using tiny lipid-coated particles called extracellular vesicles (EVs), which carry various fungal components. Previous studies found that Candida albicans EVs activate the immune response and may bridge the gap between innate and adaptive immunity. To understand this better, we investigated how these EVs activate immune cells. We demonstrated that specific components on EV surfaces, such as mannans and glucans, interact with receptors on immune cells, including Toll-like receptor 4 (TLR4) and dectin-1. Moreover, vaccinating with these EVs led to strong immune responses and full protection in mice infected with Candida. This work shows how harnessing fungal EVs might lead to effective vaccines against candidiasis.

Homologous but not heterologous COVID-19 vaccine booster elicits IgG4+ B-cells and enhanced Omicron subvariant binding

Booster vaccinations are recommended to improve protection against severe disease from SARS-CoV-2 infection. With primary vaccinations involving various adenoviral vector and mRNA-based formulations, it remains unclear if these differentially affect the immune response to booster doses. We examined the effects of homologous (mRNA/mRNA) and heterologous (adenoviral vector/mRNA) vaccination on antibody and memory B cell (Bmem) responses against ancestral and Omicron subvariants. Healthy adults who received primary BNT162b2 (mRNA) or ChAdOx1 (vector) vaccination were sampled 1-month and 6-months after their 2nd and 3rd dose (homologous or heterologous) vaccination. Recombinant spike receptor-binding domain (RBD) proteins from ancestral, Omicron BA.2 and BA.5 variants were produced for ELISA-based serology, and tetramerized for immunophenotyping of RBD-specific Bmem. Dose 3 boosters significantly increased ancestral RBD-specific plasma IgG and Bmem in both cohorts. Up to 80% of ancestral RBD-specific Bmem expressed IgG1+. IgG4+ Bmem were detectable after primary mRNA vaccination, and expanded significantly to 5–20% after dose 3, whereas heterologous boosting did not elicit IgG4+ Bmem. Recognition of Omicron BA.2 and BA.5 by ancestral RBD-specific plasma IgG increased from 20% to 60% after the 3rd dose in both cohorts. Reactivity of ancestral RBD-specific Bmem to Omicron BA.2 and BA.5 increased following a homologous booster from 40% to 60%, but not after a heterologous booster. A 3rd mRNA dose generates similarly robust serological and Bmem responses in homologous and heterologous vaccination groups. The expansion of IgG4+ Bmem after mRNA priming might result from the unique vaccine formulation or dosing schedule affecting the Bmem response duration and antibody maturation.

Immunogenicity of a Rotavirus VP8* Multivalent Subunit Vaccine in Mice.

Rotavirus remains a significant public health threat, especially in low-income countries, where it is the leading cause of severe acute childhood gastroenteritis, contributing to over 128,500 deaths annually. Although the introduction of the Rotarix and RotaTeq vaccines in 2006 marked a milestone in reducing mortality rates, approximately 83,158 preventable deaths persisted, showing ongoing challenges in vaccine accessibility and effectiveness. To address these issues, a novel subcutaneous vaccine formulation targeting multiple rotavirus genotypes has been developed. This vaccine consists of nine VP8* proteins from nine distinct rotavirus genotypes and sub-genotypes (P[4], P[6], P[8]LI, P[8]LIII, P[8]LIV, P[9], P[11], P[14], and P[25]) expressed in E. coli. Two groups of mice were immunized either with a single immunogen, the VP8* from the rotavirus Wa strain (P[8]LI), or with the nonavalent formulation. Preliminary results from mouse immunization studies showed promising outcomes, eliciting antibody responses against six of the nine immunogens. Notably, significantly higher antibody titers against VP8* P[8]LI were observed in the group immunized with the nonavalent vaccine compared to mice specifically immunized against this genotype alone. Overall, the development of parenteral vaccines targeting multiple rotavirus genotypes represents a promising strategy in mitigating the global burden of rotavirus-related morbidity and mortality, offering new avenues for disease prevention and control.

Genetic diversity and natural selection analysis of VAR2CSA and vir genes: implication for vaccine development

Variable surface antigens (VSAs) encoded by var and vir genes in Plasmodium falciparum and Plasmodium vivax, respectively, are known to be involved in malaria pathogenesis and host immune escape through antigenic variations. Knowledge of the genetic diversity of these antigens is essential for malaria control and effective vaccine development. In this study, we analysed the genetic diversity and evolutionary patterns of two fragments (DBL2X and DBL3X) of VAR2CSA gene and four vir genes (vir 4, vir 12, vir 21 and vir 27) from different endemic regions, including Southeast Asia and sub-Saharan Africa. High levels of segregating sites (S) and haplotype diversity (Hd) were observed in both var and vir genes. Among vir genes, vir 12 (S = 131, Hd = 0.996) and vir 21 (S = 171, Hd = 892) were found to be more diverse as compared to vir 4 (S = 11, Hd = 0.748) and vir 27 (S = 23, Hd = 0.814). DBL2X (S = 99, Hd = 0.996) and DBL3X (S = 307, Hd = 0.999) fragments showed higher genetic diversity. Our analysis indicates that var and vir genes are highly diverse and follow the similar evolutionary pattern globally. Some codons showed signatures of positive or negative selection pressure, but vir and var genes are likely to be under balancing selection. This study highlights the high variability of var and vir genes and underlines the need of functional experimental studies to determine the most relevant allelic forms for effective progress towards vaccine formulation and testing.

Vaccine enhancement and improved immunogenicity using erythrocytes as carriers

Achieving optimal immunogenicity and efficacy in vaccination presents significant challenges, including the need for multiple doses and the limited immunogenicity observed with certain vaccine formulations. To overcome these obstacles, researchers are exploring supplementary components and innovative delivery strategies. The development of an effective vaccine delivery technology is crucial for achieving sufficient protective immunity and disease prevention. This article focuses on the potential of erythrocytes as vaccine carriers to enhance vaccine effectiveness, particularly in the context of whole particle vaccines. The extended lifespan of erythrocytes enables sustained antigen exposure, resulting in continuous antigen presentation to immune cells. Moreover, erythrocytes exhibit biocompatibility, a well-established safety profile, and the capacity to modulate immune responses through interactions with complement receptors. These characteristics position erythrocytes as an appealing platform for targeted vaccine delivery. The hypothesis suggests utilizing bispecific monoclonal antibodies to complement receptor 1 and specific pathogen proteins to hitchhike inactivated pathogens onto erythrocytes. These modified erythrocytes can then serve as carriers to deliver the whole particle vaccine to antigen-presenting cells (APCs) or function as APCs themselves in the spleen. This article puts forward treatment protocols aimed at improving accessibility of vaccinations at the appropriate location, reducing the frequency of vaccine doses, and stimulating sufficient protective immunity.

Enhancing cross-protection against influenza by heterologous sequential immunization with mRNA LNP and protein nanoparticle vaccines

Enhancing influenza vaccine cross-protection is imperative to alleviate the significant public health burden of influenza. Heterologous sequential immunization may synergize diverse vaccine formulations and routes to improve vaccine potency and breadth. Here we investigate the effects of immunization strategies on the generation of cross-protective immune responses in female Balb/c mice, utilizing mRNA lipid nanoparticle (LNP) and protein-based PHC nanoparticle vaccines targeting influenza hemagglutinin. Our findings emphasize the crucial role of priming vaccination in shaping Th bias and immunodominance hierarchies. mRNA LNP prime favors Th1-leaning responses, while PHC prime elicits Th2-skewing responses. We demonstrate that cellular and mucosal immune responses are pivotal correlates of cross-protection against influenza. Notably, intranasal PHC immunization outperforms its intramuscular counterpart in inducing mucosal immunity and conferring cross-protection. Sequential mRNA LNP prime and intranasal PHC boost demonstrate optimal cross-protection against antigenically drifted and shifted influenza strains. Our study offers valuable insights into tailoring immunization strategies to optimize influenza vaccine effectiveness.