Immunogenicity and safety of RAZI recombinant spike protein vaccine (RCP) as a booster dose after priming with BBIBP-CorV: a parallel two groups, randomized, double blind trial.

BACKGROUND: The immunity induced by primary vaccination is effective against COVID-19; however, booster vaccines are needed to maintain vaccine-induced immunity and improve protection against emerging variants. Heterologous boosting is believed to result in more robust immune responses. This study investigated the safety and immunogenicity of the Razi Cov Pars vaccine (RCP) as a heterologous booster dose in people primed with Beijing Bio-Institute of Biological Products Coronavirus Vaccine (BBIBP-CorV). METHODS: We conducted a randomized, double-blind, active-controlled trial in adults aged 18 and over primarily vaccinated with BBIBP-CorV, an inactivated SARS-CoV-2 vaccine. Eligible participants were randomly assigned (1:1) to receive a booster dose of RCP or BBIBP-CorV vaccines. The primary outcome was neutralizing antibody activity measured by a conventional virus neutralization test (cVNT). The secondary efficacy outcomes included specific IgG antibodies against SARS-CoV-2 spike (S1 and receptor-binding domain, RBD) antigens and cell-mediated immunity. We measured humoral antibody responses at 2 weeks (in all participants) and 3 and 6 months (a subgroup of 101 participants) after the booster dose injection. The secondary safety outcomes were solicited and unsolicited immediate, local, and systemic adverse reactions. RESULTS: We recruited 483 eligible participants between December 7, 2021, and January 13, 2022. The mean age was 51.9 years, and 68.1% were men. Neutralizing antibody titers increased about 3 (geometric mean fold increase, GMFI = 2.77, 95% CI 2.26-3.39) and 21 (GMFI = 21.51, 95% CI 16.35-28.32) times compared to the baseline in the BBIBP-CorV and the RCP vaccine groups. Geometric mean ratios (GMR) and 95% CI for serum neutralizing antibody titers for RCP compared with BBIBP-CorV on days 14, 90, and 180 were 6.81 (5.32-8.72), 1.77 (1.15-2.72), and 2.37 (1.62-3.47) respectively. We observed a similar pattern for specific antibody responses against S1 and RBD. We detected a rise in gamma interferon (IFN-γ), tumor necrosis factor (TNF-α), and interleukin 2 (IL-2) following stimulation with S antigen, particularly in the RCP group, and the flow cytometry examination showed an increase in the percentage of CD3 + /CD8 + lymphocytes. RCP and BBIBP-CorV had similar safety profiles; we identified no vaccine-related or unrelated deaths. CONCLUSIONS: BBIBP-CorV and RCP vaccines as booster doses are safe and provide a strong immune response that is more robust when the RCP vaccine is used. Heterologous vaccines are preferred as booster doses. TRIAL REGISTRATION: This study was registered with the Iranian Registry of Clinical Trial at www.irct.ir , IRCT20201214049709N4. Registered 29 November 2021.

Expression and purification of TolC as a recombinant protein vaccine against Shigella flexneri and evaluation of immunogenic response in mice.

BACKGROUND: Shigella is one of the major causes of dysenteric diarrhea, which is known shigelosis. Shigelosis causes 160,000 deaths annually of diarrheal disease in the global scale especially children less than 5 years old. No licensed vaccine is available against shigelosis, therefore, efforts for develop an effective and safe vaccine against Shigella as before needed. The reverse vaccinology (RV) is a novel strategy that evaluate genome or proteome of the organism to find a new promising vaccine candidate. In this study, immunogenicity of a designed-recombinant antigen is evaluated through the in silico studies and animal experiments to predict a new immunogenic candidate against Shigella. METHODS: In the first step, proteome of Shigella flexneri was obtained from UniProtKB and then the outer membrane and extracellular proteins were predicted. In this study TolC as an outer membrane protein was selected and confirmed among candidates. In next steps, pre-selected protein was evaluated for transmembrane domains, homology, conservation, antigenicity, solubility, and B- and T-cell prediction by different online servers. RESULT: TolC as a conserved outer membrane protein, using different immune-informatics tools had acceptable scores and was selected as the immunogenic antigen for animal experiment studies. Recombinant TolC protein after expression and purification, was administered to BALB/c mice over three intraperitoneal routes. The sera of mice was used to evaluate the IgG1 production assay by indirect-ELISA. The immunized mice depicted effective protection against 2LD50 of Shigella. Flexneri ATCC12022 (challenge study). CONCLUSION: Therefore, the reverse vaccinology approach and experimental test results demonstrated that TolC as a novel effective and immunogenic antigen is capable for protection against shigellosis.

Evaluation of long-term immune response in cattle to botulism using a recombinant E. coli bacterin formulated with Montanide™ ISA 50 and aluminum hydroxide adjuvants.

Botulism is a severe disease caused by potent botulinum neurotoxins (BoNTs) produced by Clostridium botulinum. This disease is associated with high-lethality outbreaks in cattle, which have been linked to the ingestion of preformed BoNT serotypes C and D, emphasizing the need for effective vaccines. The potency of current commercial toxoids (formaldehyde-inactivated BoNTs) is assured through tests in guinea pigs according to government regulatory guidelines, but their short-term immunity raises concerns. Recombinant vaccines containing the receptor-binding domain have demonstrated potential for eliciting robust protective immunity. Previous studies have demonstrated the safety and effectiveness of recombinant E. coli bacterin, eliciting high titers of neutralizing antibodies against C. botulinum and C. perfringens in target animal species. In this study, neutralizing antibody titers in cattle and the long-term immune response against BoNT/C and D were used to assess the efficacy of the oil-based adjuvant compared with that of the aluminum hydroxide adjuvant in cattle. The vaccine formulation containing Montanide™ ISA 50 yielded significantly higher titers of neutralizing antibody against BoNT/C and D (8.64 IU/mL and 9.6 IU/mL, respectively) and induced an immune response that lasted longer than the response induced by aluminum, extending between 30 and 60 days. This approach represents a straightforward, cost-effective strategy for recombinant E. coli bacterin, enhancing both the magnitude and duration of the immune response to botulism.

Comparative protective efficacy of a newly generated live recombinant thermostable highly attenuated vaccine rK148/GVII-F using a single regimen against lethal NDV GVII.1.1.

RESEARCH HIGHLIGHTS: A thermostable, safe, and effective NDV GVII recombinant vaccine was generated.Fusion gene replacement with GVII did not affect GI K148/08 virus thermostability.Strain rK148/GVII-F provided adequate protection against a lethal NDV challenge.Oropharyngeal shedding was significantly reduced on post-challenge days 5 and 7.

Prevention of nodules and enhancement of antibody response to genetically engineered recombinant vaccine against Human Chorionic Gonadotropin (hCG) for contraception.

OBJECTIVE: Human Chorionic Gonadotropin (hCG) plays a crucial role in embryo implantation and in maintenance of pregnancy. An immuno-contraceptive approach involves the use of a recombinant hCGß-LTB vaccine formulated with adjuvant Mycobacterium indicus pranii (MIP), to prevent pregnancy without disturbing ovulation, hormonal profiles, and menstrual cycles in women. The present work in mice was designed to address issues encountered in clinical trials conducted with hCGß-LTB vaccine, with focus on two primary concerns. Firstly, it aimed to determine the optimal vaccine dosage required to induce a high level of anti-hCG antibodies. Secondly, it aimed to assess the safety profile of the vaccine, specifically injection site reactions in the form of nodules, observed in some of the subjects. METHODS AND RESULTS: Studies undertaken indicate that a 2 µg dose of the protein version of the vaccine, administered in mice through the intramuscular route, can induce high anti-hCG titres. Furthermore, administering a booster dose enhances the antibody response. Our findings suggest that the concentration and frequency of administration of the adjuvant MIP can also be reduced without compromising vaccine efficacy. CONCLUSION: The issue of nodule formation at the injection site can be mitigated either by administering the vaccine along with MIP intramuscularly or injecting hCG vaccine and MIP at separate intradermal sites. Thus, protein vaccine administered at a 2µg dose via the intramuscular route addresses both efficacy and safety concerns.

The Phase I/II clinical trials initiated with the recombinant hCG vaccine in women revealed inadequate antibody titres in all subjects, alongside the development of nodules at the injection sites in some participants. Studies were undertaken in mice to propose potential strategies for mitigating injection site reactions and enhancing the antibody response. It was concluded that the optimum dose of the protein version of the vaccine to get high antibody titres, is 2 µg administered intramuscularly while upholding safety standards.

Reduced Respiratory Syncytial Virus Load, Symptoms, and Infections: A Human Challenge Trial of MVA-BN-RSV Vaccine.

BACKGROUND: Respiratory syncytial virus (RSV) causes significant disease burden in older adults. MVA-BN-RSV is a novel poxvirus-vectored vaccine encoding internal and external RSV proteins. METHODS: In a phase 2a randomized double-blind, placebo-controlled trial, healthy participants aged 18 to 50 years received MVA-BN-RSV or placebo, then were challenged 4 weeks later with RSV-A Memphis 37b. Viral load was assessed from nasal washes. RSV symptoms were collected. Antibody titers and cellular markers were assessed before and after vaccination and challenge. RESULTS: After receiving MVA-BN-RSV or placebo, 31 and 32 participants, respectively, were challenged. Viral load areas under the curve from nasal washes were lower (P = .017) for MVA-BN-RSV (median = 0.00) than placebo (median = 49.05). Total symptom scores also were lower (median = 2.50 and 27.00, respectively; P = .004). Vaccine efficacy against symptomatic, laboratory-confirmed or culture-confirmed infection was 79.3% to 88.5% (P = .022 and .013). Serum immunoglobulin A and G titers increased approximately 4-fold after MVA-BN-RSV vaccination. Interferon-γ-producing cells increased 4- to 6-fold after MVA-BN-RSV in response to stimulation with the encoded RSV internal antigens. Injection site pain occurred more frequently with MVA-BN-RSV. No serious adverse events were attributed to vaccination. CONCLUSIONS: MVA-BN-RSV vaccination resulted in lower viral load and symptom scores, fewer confirmed infections, and induced humoral and cellular responses. CLINICAL TRIALS REGISTRATION: NCT04752644.

Protective efficacy and correlates of immunity of immunodominant recombinant Babesia microti antigens.

Babesia microti, an intraerythrocytic apicomplexan parasite, is the primary causative agent of human babesiosis and an emerging threat to public health in the United States and elsewhere. An effective vaccine against B. microti would reduce disease severity in acute babesiosis patients and shorten the parasitemic period in asymptomatic individuals, thereby minimizing the risk of transfusion-transmitted babesiosis. Here we report on immunogenicity, protective efficacy, and correlates of immunity following immunization with four immunodominant recombinantly produced B. microti antigens-Serine Reactive Antigen 1 (SERA1), Maltese Cross Form Related Protein 1 (MCFRP1), Piroplasm ß-Strand Domain 1 (PißS1), and Babesia microti Alpha Helical Cell Surface Protein 1 (BAHCS1)-delivered subcutaneously in Montanide ISA 51/CpG adjuvant in three doses to BALB/c mice. Following B. microti parasite challenge, BAHCS1 led to the highest reduction in peak parasitemia (67.8%), followed by SERA1 (44.8%) and MCFRP1 (41.9%); PißS1 (27.6%) had minimal protective effect. All four B. microti antigens induced high ELISA total IgG and each isotype; however, antibody levels did not directly correlate with anti-parasitic activity in mice. Increased prechallenge levels of some cell populations including follicular helper T cells (TFH) and memory B cells, along with a set of six cytokines [IL-1α, IL-2, IL-3, IL-6, IL-12(p40), and G-CSF] that belong to both innate and adaptive immune responses, were generally associated with protective immunity. Our results indicate that mechanisms driving recombinant B. microti antigen-induced immunity are complex and multifactorial. We think that BAHCS1 warrants further evaluation in preclinical studies.

Efficacy of recombinant H5 vaccines delivered in ovo or day of age in commercial broilers against the 2015 U.S. H5N2 clade 2.3.4.4c highly pathogenic avian Influenza virus.

BACKGROUND: Avian influenza is a highly contagious, agriculturally relevant disease that can severely affect the poultry industry and food supply. Eurasian-origin H5Nx highly pathogenic avian influenza viruses (HPAIV) (clade 2.3.4.4) have been circulating globally in wild birds with spill over into commercial poultry operations. The negative impact to commercial poultry renewed interest in the development of vaccines against these viruses to control outbreaks in the U.S. METHODS: The efficacy of three recombinant H5 vaccines delivered in ovo or day of age were evaluated in commercial broilers challenged with the 2015 U.S. H5N2 clade 2.3.4.4c HPAIV. The recombinant vaccines included an alphavirus RNA particle vaccine (RP-H5), an inactivated reverse genetics-derived (RG-H5) and recombinant HVT vaccine (rHVT-AI) expressing H5 hemagglutinin (HA) genes. In the first experiment, in ovo vaccination with RP-H5 or rHVT-AI was tested against HPAI challenge at 3 or 6 weeks of age. In a second experiment, broilers were vaccinated at 1 day of age with a dose of either 107 or 108 RP-H5, or RG-H5 (512 HA units (HAU) per dose). RESULTS: In experiment one, the RP-H5 provided no protection following in ovo application, and shedding titers were similar to sham vaccinated birds. However, when the RP-H5 was delivered in ovo with a boost at 3 weeks, 95% protection was demonstrated at 6 weeks of age. The rHVT-AI vaccine demonstrated 95 and 100% protection at 3 and 6 weeks of age, respectively, of challenged broilers with reduced virus shedding compared to sham vaccinated birds. Finally, when the RP-H5 and rHVT vaccines were co-administered at one day of age, 95% protection was demonstrated with challenge at either 3 or 6 weeks age. In the second experiment, the highest protection (92%) was observed in the 108 RP-H5 vaccinated group. Significant reductions (p < 0.05) in virus shedding were observed in groups of vaccinated birds that were protected from challenge. The RG-H5 provided 62% protection from challenge. In all groups of surviving birds, antibody titers increased following challenge. CONCLUSIONS: Overall, these results demonstrated several strategies that could be considered to protected broiler chickens during a H5 HPAI challenge.

Intranasal administration with recombinant vaccine PRVXJ-delgE/gI/TK-S induces strong intestinal mucosal immune responses against PDCoV.

Porcine deltacoronavirus (PDCoV) is a novel coronavirus that causes enteric diseases in pigs leading to substantial financial losses within the industry. The absence of commercial vaccines and limited research on PDCoV vaccines presents significant challenges. Therefore, we evaluated the safety and immunogenicity of recombinant pseudorabies virus (PRV) rPRVXJ-delgE/gI/TK-S through intranasal mucosal immunization in weaned piglets and SPF mice. Results indicated that rPRVXJ-delgE/gI/TK-S safely induced PDCoV S-specific and PRV gB-specific antibodies in piglets, with levels increasing 7 days after immunization. Virus challenge tests demonstrated that rPRVXJ-delgE/gI/TK-S effectively improved piglet survival rates, reduced virus shedding, and alleviated clinical symptoms and pathological damage. Notably, the recombinant virus reduced anti-inflammatory and pro-inflammatory responses by regulating IFN-γ, TNF-α, and IL-1ß secretion after infection. Additionally, rPRVXJ-delgE/gI/TK-S colonized target intestinal segments infected with PDCoV, stimulated the secretion of cytokines by MLVS in mice, stimulated sIgA secretion in different intestinal segments of mice, and improved mucosal immune function. HE and AB/PAS staining confirmed a more complete intestinal mucosal barrier and a significant increase in goblet cell numbers after immunization. In conclusion, rPRVXJ-delgE/gI/TK-S exhibits good immunogenicity and safety in mice and piglets, making it a promising candidate vaccine for PDCoV.

Confirming the absence of parental African swine fever virus as a potential contaminant of recombinant live attenuated ASF vaccines.

African swine fever (ASF) is a devastating disease that is currently producing a panzootic significantly impacting the swine industry worldwide. One of the major challenges for advancing the development of ASF vaccines has been the absence of international standards for ASF vaccine purity, potency, safety, and efficacy. To date, the most effective experimental vaccines have been live attenuated strains of viruses. Most of these promising vaccine candidates have been developed by deleting virus genes involved in the process of viral pathogenesis and disease production. This approach requires genomic modification of a parental virus field strain through a process of homologous recombination followed by purification of the recombinant attenuated virus. In this scenario, it is critical to confirm the absence of any parental virulent virus in the final virus stock used for vaccine production. We present here a protocol to establish the purity of virus stock using the live attenuated vaccine candidates ASFV-G-ΔMGF, ASFV-G-Δ9 GLΔUK and ASFV-G-ΔI177L. Procedures described here includes inoculation in susceptible pigs followed by the assessment of the obtained material by differential qPCRs that allows the identification of vaccine virus from ASFV field isolates. This protocol is proposed as a model to ensure that master seed virus stock used for vaccine production does not contain residual parental virulent virus. Procedures described here includes a passage in susceptible pigs followed by the assessment of the obtained material by differential qPCRs that allows the identification of vaccine virus from ASFV field isolates.

Engineering an Optimal Y280-Lineage H9N2 Vaccine Strain by Tuning PB2 Activity.

H9N2 avian influenza A viruses (AIVs) cause economic losses in the poultry industry and provide internal genomic segments for the evolution of H5N1 and H7N9 AIVs into more detrimental strains for poultry and humans. In addition to the endemic Y439/Korea-lineage H9N2 viruses, the Y280-lineage spread to Korea since 2020. Conventional recombinant H9N2 vaccine strains, which bear mammalian pathogenic internal genomes of the PR8 strain, are pathogenic in BALB/c mice. To reduce the mammalian pathogenicity of the vaccine strains, the PR8 PB2 was replaced with the non-pathogenic and highly productive PB2 of the H9N2 vaccine strain 01310CE20. However, the 01310CE20 PB2 did not coordinate well with the hemagglutinin (HA) and neuraminidase (NA) of the Korean Y280-lineage strain, resulting in a 10-fold lower virus titer compared to the PR8 PB2. To increase the virus titer, the 01310CE20 PB2 was mutated (I66M-I109V-I133V) to enhance the polymerase trimer integrity with PB1 and PA, which restored the decreased virus titer without causing mouse pathogenicity. The reverse mutation (L226Q) of HA, which was believed to decrease mammalian pathogenicity by reducing mammalian receptor affinity, was verified to increase mouse pathogenicity and change antigenicity. The monovalent Y280-lineage oil emulsion vaccine produced high antibody titers for homologous antigens but undetectable titers for heterologous (Y439/Korea-lineage) antigens. However, this defect was corrected by the bivalent vaccine. Therefore, the balance of polymerase and HA/NA activities can be achieved by fine-tuning PB2 activity, and a bivalent vaccine may be more effective in controlling concurrent H9N2 viruses with different antigenicities.

In silico design of recombinant multi-epitope vaccine against influenza A virus.

BACKGROUND: Influenza A virus is one of the leading causes of annual mortality. The emerging of novel escape variants of the influenza A virus is still a considerable challenge in the annual process of vaccine production. The evolution of vaccines ranks among the most critical successes in medicine and has eradicated numerous infectious diseases. Recently, multi-epitope vaccines, which are based on the selection of epitopes, have been increasingly investigated. RESULTS: This study utilized an immunoinformatic approach to design a recombinant multi-epitope vaccine based on a highly conserved epitope of hemagglutinin, neuraminidase, and membrane matrix proteins with fewer changes or mutate over time. The potential B cells, cytotoxic T lymphocytes (CTL), and CD4 T cell epitopes were identified. The recombinant multi-epitope vaccine was designed using specific linkers and a proper adjuvant. Moreover, some bioinformatics online servers and datasets were used to evaluate the immunogenicity and chemical properties of selected epitopes. In addition, Universal Immune System Simulator (UISS) in silico trial computational framework was run after influenza exposure and recombinant multi-epitope vaccine administration, showing a good immune response in terms of immunoglobulins of class G (IgG), T Helper 1 cells (TH1), epithelial cells (EP) and interferon gamma (IFN-g) levels. Furthermore, after a reverse translation (i.e., convertion of amino acid sequence to nucleotide one) and codon optimization phase, the optimized sequence was placed between the two EcoRV/MscI restriction sites in the PET32a+ vector. CONCLUSIONS: The proposed "Recombinant multi-epitope vaccine" was predicted with unique and acceptable immunological properties. This recombinant multi-epitope vaccine can be successfully expressed in the prokaryotic system and accepted for immunogenicity studies against the influenza virus at the in silico level. The multi-epitope vaccine was then tested with the Universal Immune System Simulator (UISS) in silico trial platform. It revealed slight immune protection against the influenza virus, shedding the light that a multistep bioinformatics approach including molecular and cellular level is mandatory to avoid inappropriate vaccine efficacy predictions.

Seroresponse to Inactivated and Recombinant Influenza Vaccines Among Maintenance Hemodialysis Patients.

RATIONALE & OBJECTIVE: High-dose influenza vaccine provides better protection against influenza infection in older adults than standard-dose vaccine. We compared vaccine seroresponse among hemodialysis patients over a period of 4 months after administration of high-dose trivalent inactivated (HD-IIV3), standard-dose quadrivalent inactivated (SD-IIV4), or quadrivalent recombinant quadrivalent (RIV4) influenza vaccine. STUDY DESIGN: Prospective observational study. SETTING & PARTICIPANTS: Patients at 4 hemodialysis clinics who received influenza vaccine. EXPOSURE: Type of influenza vaccine. OUTCOME: Hemagglutination inhibition (HI) titers were measured at baseline and at 1, 2, 3, and 4 months after vaccination. The primary outcome was seroprotection rates at HI titers of at least 1:40 and at least 1:160 (antibody levels providing protection from infection in approximately 50% and 95% of immunocompetent individuals, respectively) at 1, 2, 3, and 4 months after vaccination. ANALYTICAL APPROACH: We calculated geometric mean titer as well as seroprotection and seroconversion rates. Adjusted generalized linear models with additional trend analyses were performed to evaluate the association between vaccine type and outcomes. RESULTS: 254 hemodialysis patients were vaccinated against influenza with HD-IIV3 (n = 141), SD-IIV4 (n = 36), or RIV4 (n = 77). A robust initial seroresponse to influenza A strains was observed after all 3 vaccines. Geometric mean titer and seroprotection (HI titer ≥1:160) rates against influenza A strains were higher and more sustained with HD-IIV3 than SD-IIV4 or RIV4. More than 80% of patients vaccinated with HD-IIV3 were seroprotected (HI titer ≥1:160) at month 4 (P < 0.001), whereas, among patients vaccinated with SD-IIV4 or RIV4, seroprotection rates were similar to those at baseline. Seroprotection rates were lower against B strains for all vaccines. LIMITATIONS: Because of the use of observational data, bias from unmeasured confounders may exist. Some age subgroups were small in number. Clinical outcome data were not available. CONCLUSIONS: Hemodialysis patients exhibited high seroprotection rates after all 3 influenza vaccines. The seroresponse waned more slowly with HD-IIV3 compared with SD-IIV4 and RIV4 vaccines.

DnaJ, a promising vaccine candidate against Ureaplasma urealyticum infection.

Ureaplasma urealyticum (U. urealyticum, Uu) is a common sexually transmitted pathogen that is responsible for diseases such as non-gonococcal urethritis, chorioamnionitis, and neonatal respiratory diseases. The rapid emergence of multidrug-resistant bacteria threatens the effective treatment of Uu infections. Considering this, vaccination could be an efficacious medical intervention to prevent Uu infection and disease. As a highly conserved molecular chaperone, DnaJ is expressed and upregulated by pathogens soon after infection. Here, we assessed the vaccine potential of recombinant Uu-DnaJ in a mouse model and dendritic cells. Results showed that intramuscular administration of DnaJ induced robust humoral- and T helper (Th) 1 cell-mediated immune responses and protected against genital tract infection, inflammation, and the pathologic sequelae after Uu infection. Importantly, the DnaJ protein also induced the maturation of mouse bone marrow-derived dendritic cells (BMDCs), ultimately promoting naïve T cell differentiation toward the Th1 phenotype. In addition, adoptive immunization of DnaJ-pulsed BMDCs elicited antigen-specific Immunoglobulin G2 (IgG2) antibodies as well as a Th1-biased cellular response in mice. These results support DnaJ as a promising vaccine candidate to control Uu infections. KEY POINTS: • A novel recombinant vaccine was constructed against U. urealyticum infection. • Antigen-specific humoral and cellular immune responses after DnaJ vaccination. • Dendritic cells are activated by Uu-DnaJ, which results in a Th1-biased immune response.

Recent progress and advances towards developing enterovirus 71 vaccines for effective protection against human hand, foot and mouth disease (HFMD).

The main pathogen causing severe and neurotrophic hand, foot and mouth disease (HFMD) is enterovirus A71 (EV71). EV71 infection is among the major cause of serious public health burden and economic loss especially in the Asia-pacific region. Yet, no specific anti-viral treatment against this life-threatening infection is currently available. Thus, the best way to control EV71 infection is by vaccination with an effective and safe vaccine. Several strategies are being employed to develop vaccines against EV71. These include conventional and modern recombinant vaccine strategies. Conventional vaccines such as inactivated EV71 vaccines are the most studied and advanced vaccines against HFMD. Recombinant HFMD vaccines developed based on the recombinant DNA technology have been employed but are mostly at early or late preclinical development stage. In this article, we discuss the recent progress and advances in modern recombinant strategies of EV71 vaccine development including subunit, VLP, epitope-based, DNA, and vector-based vaccines, as well as conventional approaches, focusing on their various prospects, advantages and disadvantages.

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.

Impact of Human Papillomavirus Vaccination, Rwanda and Bhutan.

Rwanda and Bhutan, 2 low- and middle-income countries, implemented primarily school-based national human papillomavirus (HPV) vaccination in 2011 (Rwanda) and 2010 (Bhutan). We estimated vaccination effectiveness through urine-based HPV prevalence surveys in schools in 2013-2014 and 2017. In Rwanda, 912 participants from baseline surveys and 1,087 from repeat surveys were included, and in Bhutan, 973 participants from baseline surveys and 909 from repeat surveys were included. The overall effectiveness against vaccine-targeted HPV types (i.e., HPV-6/11/16/18) was 78% (95% CI 51%-90%) in Rwanda, and 88% (6%-99%) in Bhutan and against other α-9 types was 58% (21-78) in Rwanda and 63% (27-82) in Bhutan. No effect against other HPV types was detectable. Prevalence of vaccine-targeted HPV types decreased significantly, as well as that of other α-9 types, suggesting cross-protection. These findings provide direct evidence from low- and middle-income countries of the marked effectiveness of high-coverage school-based, national HPV vaccination programs.

In Silico designing and immunogenic production of the multimeric CfaB*ST, CfaE, LTB antigen as a peptide vaccine against Enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) is the most frequent bacterial cause of diarrhea particularly reported in children of developing countries and also travelers. Enterotoxins and colonization factor antigens (CFAs) are two major virulence factors in ETEC pathogenesis. Colonization factor antigen I (CFA/I) includes major pilin subunit CfaB, and a minor adhesive subunit (CfaE), and enterotoxins consisting of heat-labile toxin subunit B (LTB) and heat-stable toxin (ST). Chimeric proteins (CCL) carrying epitopes and adjuvant sequences increase the possibility of eliciting a broad cellular or effective immune response. In the present study, a chimeric candidate vaccine containing CfaB*ST, CfaE, and LTB (CCL) was designed via in silico techniques. This chimeric gene was synthesized by using codon usage of E. coli for increasing the expression of the recombinant protein. After designing the chimeric construct, it showed a high antigenicity index estimated by the vaxiJen server. Linear and conformational B-cell epitopes were identified and indicated suitable immunogenicity of this multimeric recombinant protein. Thermodynamic analyses for mRNA structures revealed the appropriate folding of the RNA representative good stability of this molecule. In silico scanning was done to predict the 3D structure of the protein, and modeling was validated using the Ramachandran plot analysis. The chimeric protein (rCCL) was expressed in a prokaryotic expression system (E. coli), purified, and analyzed for their immunogenic properties. It was revealed that the production of a high titer of antibody produced in immunized mice could neutralize the ETEC using the rabbit ileal loop tests. The results indicated that the protein inferred from the recombinant protein (rCCL) construct could act as a proper vaccine candidate against three critical causative agents of diarrheal bacteria at the same time.

Design and development of a simple method for the detection and quantification of residual host cell DNA in recombinant rotavirus vaccine.

Rotavirus recombinant vaccine is usually produced in Vero cells. Residual host DNA may reside in the final product and is considered a source of contamination. WHO protocols indicate that biological products should be free of any type of impurity such as nucleic acids, endotoxins, and host cell intermediate materials. Therefore, all recombinant biological therapeutics should be assessed for residual host DNA. In the present study, a sensitive and specific real-time PCR method was developed to detect residual host cell DNA in the final product. The Beta-actin gene of Vero cells was selected to detect residual host cell DNA. One set of primers and a TaqMan probe were designed for the gene using AlleleID 6 software. Real-time PCR reactions were set up, and efficiency of 84% was obtained. The sensitivity and limit of detection of the assay were determined to be 0.176 Fg/µl and 0.044 Fg/µl, respectively. The intra-assay and inter-assay variations were 4.4% and 1.04%, respectively. Furthermore, the specificity and sensitivity of the assay were high enough, and the detection limit was lower than that of the FDA and WHO standards. This indicates that our assay is highly specific and sensitive to detect residual host DNA of Vero cells in the recombinant rotavirus vaccine.

Protective efficacy of four heat-shock proteins as recombinant vaccines against photobacteriosis in Asian seabass (Lates calcarifer).

Photobacterium damselae subsp. piscicida (Phdp) is the causative agent of photobacteriosis in marine fish and is responsible for huge losses to marine aquaculture worldwide. Efforts have been made to develop a vaccine against this disease. Heat-shock proteins (HSPs) are a family of proteins that are ubiquitous in cellular life. Bacteria produce elevated levels of HSPs as a survival strategy when exposed to stressful environments in a host during infection. This group of proteins are also important antigens that can induce both humoral and cellular immune responses. In this study, four HSPs of Phdp, HSP90, HSP33, HSP70, and DnaJ, were selected for cloning and recombinant expression. Western blotting with rabbit anti-Phdp helped identify rHSP70 and rHSP33 as immunogenic proteins. Asian seabass (Lates calcarifer) immunised with rHSP90, rHSP33, rHSP70, and rDnaJ showed 48.28%, 62.07%, 51.72%, and 31.03% relative percent survival, respectively, after being challenged with Phdp strain AOD105021. High expression levels of immune-related genes and high antibody titres were observed in the rHSP33 group, and the sera of this group also exhibited a high level of bactericidal activity against Phdp. Collectively, our results suggest that HSP33 is a potential candidate for vaccine development against Phdp infection.