Background: The combined diphtheria–tetanus–acellular pertussis (three-component), Haemophilus influenzae type b (Hib, conjugate), and ACYW135 meningococcal (conjugate) vaccine (DTaP-Hib-MCV4) offers a promising alternative to single-component vaccines, potentially simplifying immunization schedules and improving vaccination coverage. Methods: We evaluated the safety, immunogenicity, and protective efficacy of DTaP-Hib-MCV4 in animal models. Acute and long-term toxicity studies were conducted in Sprague-Dawley (SD) rats with equal numbers of male and female animals. Immunogenicity was assessed in female NIH mice and SD rats using a three-dose regimen at 14-day intervals. Orbital blood was collected 14 days post-immunization to measure IgG titers against pertussis, diphtheria, tetanus, Hib, and meningococcal antigens. The protective efficacy was determined using potency tests for the pertussis, diphtheria, and tetanus components; passive protection studies for Hib; and serum bactericidal antibody (SBA) titers against A/C/Y/W135 meningococcal serogroups. Results: Acute and repeated-dose toxicity studies in SD rats showed no signs of abnormal toxicity or irritation at either high (three doses/rat) or low (one dose/rat) doses levels. The no-observed-adverse-effect level (NOAEL) for DTaP-Hib-MCV4 was established at three doses/rat after 8 weeks of repeated intramuscular administration and a 4-week recovery period. Specific IgG antibodies against all the vaccine components were detected in animal sera at both one and three doses/rat, with no evidence of immunotoxicity. Following two-dose primary immunization in murine models, the combined vaccine elicited robust antigen-specific antibody responses, with geometric mean titers (GMTs) as follows: 1,280,000 for pertussis toxin (PT); 761,093 for filamentous hemagglutinin (FHA); 1,159,326 for pertactin (PRN); 1,659,955 for diphtheria toxoid (DT); 1,522,185 for tetanus toxoid (TT); 99 for Haemophilus influenzae type b (Hib); and 25,600, 33,199, 8300, and 9051 for serogroups A, C, Y, and W135 of Neisseria meningitidis, respectively. In the rat models, three-dose primary immunization also elicited robust antigen-specific antibody responses. Protection studies demonstrated efficacy against pertussis, tetanus toxin, and diphtheria toxin challenges. In the Hib challenge study, none of the 10 animals given anti-DTaP-Hib-MCV4 antiserum developed bacteremia after the live Hib challenge (vs. 5814/0.1 mL in the negative control, p < 0.001). In addition, the SBA titers against meningococcal serogroups exceeded the protective threshold (≥1:8) in 92.2% of the immunized mice and 100% of the immunized rats. Crucially, the combined vaccine induced potent immune responses and protective efficacy, with antibody levels and protection against each component antigen comparable to or greater than those of the individual components: DTaP, Hib, and MCV4. Conclusions: These findings demonstrate that the DTaP-Hib-MCV4 combined vaccine is both safe and immunogenic, supporting its potential as a viable alternative to individual vaccines. This combined vaccine may streamline immunization programs and enhance vaccination coverage.

Pertussis remains a leading cause of infant mortality globally, with rising cases in China post-COVID-19. Despite vaccination, waning immunity from acellular pertussis vaccines has driven resurgence. We evaluated a novel diphtheria, tetanus and pertussis (acellular, three components) combined vaccine, adsorbed (DTcP), China's first genetically engineered three-component pertussis vaccine, administered under two primary schedules (2/3/4 vs. 2/4/6 months) compared to licensed DTaP-IPV-Hib (Pentaxim). In this randomized, blinded, phase III clinical trial, 1380 healthy 2-month-old infants from Henan, China, received DTcP-1 (2/3/4 months) or DTcP-2 (2/4/6 months) or DTaP-IPV-Hib (2/3/4 months). The primary safety endpoints were the incidence of adverse reactions within 0∼30 days after primary vaccination. The primary immunogenicity endpoints were to evaluate the non-inferiority and superiority of seroconversion rates and geometric mean concentrations (GMCs) of anti-pertussis toxoid (PT), filamentous hemagglutinin (FHA), pertactin (PRN), diphtheria toxoid (DT), tetanus toxoid (TT), antibodies 30 days after primary vaccination. Immunogenicity was assessed via Luminex-based multiplex immunoassay. Both DTcP-1 and DTcP-2 schedules demonstrated non-inferior safety to DTaP-IPV-Hib (total adverse reactions: 14.52/16.59% vs. 16.91%, P = 0.183), with DTcP-2 (2/4/6 months) showing lower swelling (2.59% vs. 4.83%, P = 0.008) and irritability (0.07% vs. 1.02/1.40%, P < 0.001). DTcP-2 elicited higher GMCs against pertussis antigens (PT: 84.23 vs. 65.35; FHA: 132.16 vs. 102.13, both P < 0.001) and comparable DT/TT responses. DTcP exhibited favorable safety and superior pertussis immunogenicity, particularly with the 2/4/6-month schedule. Its genetically engineered three-component design offers a promising strategy to combat pertussis amid global resurgence.

Traditional polysaccharide vaccines are constrained by streptococcus pneumoniae diversity. We propose a protein-based pneumococcal vaccine (PBPV) — formulated with conserved surface proteins P3296, P5668, PRx1, and pneumolysin (Ply) — that could potentially offer superior immune breadth independent of capsular polysaccharide serotypes. Here, we evaluated the multifunctional antibody responses induced by PBPV, including immunogenicity, Ply neutralization, opsonophagocytic activity (OPA), and such nonopsonic functions as NK cell activation (ADNKA), antibody-dependent cellular phagocytosis, and neutrophil phagocytosis (ADNP) in a cohort of 50- to 69-year-olds. While PBPV showed shorter-lasting immune responses, including reduced Ply-neutralizing capacity, it provided broader cross-serotype protection than 23-valent pneumococcal polysaccharide vaccine. Correlation analysis identified distinct PspA-specific IgG subclass roles: P3296-IgG1 correlated with OPA, and IgG3 correlated with ADNKA/ADNP; P5668-IgG2 correlated with ADNKA/ADNP, and IgG3 correlated with OPA; and PRx1-IgG2 correlated with OPA, and IgG3 correlated with ADNKA. Critically, while no efficacy data have yet confirmed the protective effect of PBPV, its targeting of conserved proteins rather than capsular polysaccharides enables simplified manufacturing and expanded coverage, positioning it as a promising alternative to traditional multipolysaccharide vaccines.

Background/Objectives: Varicella-zoster virus (VZV) causes herpes zoster (HZ/shingles), particularly in older adults with weakened cell-mediated immunity (CMI), which is essential for controlling VZV reactivation and reducing HZ severity. Currently vaccines, like recombinant subunit or live-attenuated vaccine, showed shortcomings in eliciting CD8⁺ T-cell responses. Addressing this, we utilized the novel replication-defective chimpanzee adenovirus vector ChAdOx1 to construct the ChAdOx1-VZV (CVE) vaccine, using full-length glycoprotein E (gE) as antigen. This study evaluated the immunogenicity of a heterologous intramuscular (IM) prime/intranasal (IN) boost regimen with the aim of developing a novel VZV vaccine candidate. Methods: BALB/c mice were immunized with CVE using homologous or heterologous prime-boost regimens via IM or IN. And cynomolgus macaques were immunized intramuscularly with three doses of CVE. Cellular responses were assessed by intracellular cytokine staining (ICS) and IFN-γ ELISpot using splenocytes and PBMCs. Humoral responses were evaluated by serum gE-IgG ELISA and bone-marrow LLPC ELISpot. Memory subsets and tissue-resident T cells were analyzed by flow cytometry. Results: Heterologous IM prime/IN boost CVE regimen markedly enhanced both cellular and humoral responses, especially CD8⁺ T-cell responses. The induced LLPC and memory T cell responses indicate the potential for long-term protection against herpes zoster. In cynomolgus macaques, CVE induced robust serum gE-specific IgG responses and strong IFN-γ secreting T-cell activity, supporting the immunogenicity of CVE in a genetically distinct primate model and enhancing its clinical translational potential. Conclusions: CVE induces potent cellular and humoral immunogenicity, with IM prime/IN boost vaccination. Cross species immunogenicity observed in nonhuman primates further strengthens the translational relevance of this platform. These findings support CVE as a promising herpes zoster vaccine candidate and provide a rationale for continued evaluation in human-relevant systems.

Background: Pneumococcal diseases remain a global threat due to the serotype-specific limitations of polysaccharide vaccines. This study evaluated a recombinant protein-based pneumococcal vaccine (PBPV) combining three PspA variants (PRX1/Family1Clade2, P3296/Family2/Clade3, P5668/Family2/Clade4) and detoxified pneumolysin (PlyLD). PspA targets conserved surface epitopes to block immune evasion and achieve broad coverage, while PlyLD neutralizes pore-forming toxins and enhances adaptive immunity. Methods: We evaluated the safety and immunogenicity of the PBPV in animal models. Acute toxicity studies were conducted by administering a single intramuscular injection to ICR mice, whereas chronic toxicity and immunogenicity studies were performed in cynomolgus monkeys via repeated intramuscular injections, with an equal number of male and female animals in both groups. Immune responses were assessed using ELISA, multiplexed opsonophagocytic killing assays (MOPAs), and neutralizing antibody assays. Results: Acute toxicity studies in ICR mice showed no signs of abnormal toxicity or irritation at one-dose levels. In the chronic toxicity study, cynomolgus monkeys received repeated intramuscular injections once every 3 weeks for a total of four administrations, at doses of one dose/monkey and five doses/monkey, followed by a 4-week recovery period. No significant systemic toxic reactions were observed, and the safe dose was determined to be five doses/monkey. In the immunogenicity study of monkey serum, both low-dose and high-dose groups demonstrated significant increases in antigen-specific IgG titers against each component; opsonophagocytic killing activity against pneumococcal strains from Clades 2, 3, and 4 from PspA Families 1 and 2; and neutralization antibody titers against pneumolysin post-vaccination. Conclusions: The recombinant protein-based pneumococcal vaccine exhibited a favorable safety profile and potent immunogenicity in animal models, indicating promise for broad protection against pneumococcal disease. These findings support the further development of PBPVs as a viable alternative to conventional polysaccharide-based vaccines.

Background: Streptococcus pneumoniae (Spn) is a leading bacterial pathogen responsible for severe invasive diseases, including meningitis, sepsis, and pneumonia. Current pneumococcal vaccines, which are all based on capsular polysaccharide antigens, provide limited protection and are further compromised by post-vaccination serotype replacement. Pneumococcal surface protein A (PspA), a highly conserved virulence factor expressed across diverse serotypes, has emerged as a promising candidate antigen for novel protein-based vaccines. However, progress in this field has been hindered by the absence of standardized in vitro functional antibody assays. Methods: This study established a robust functional antibody detection method for PspA-based protein vaccines by modifying the conventional multiplex opsonophagocytic killing assay (MOPA), originally designed for polysaccharide-based vaccines. Using polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) typing, a target strain panel was selected and developed to include representative strains from PspA Family 1-Clade 2 and Family 2-Clades 3 and 4. The MOPA protocol was optimized by extending the phagocytic reaction time to enhance sensitivity. Specificity was confirmed through recombinant PspA competitive inhibition assays. Results: The assay demonstrated high linearity (R² ≥ 0.98) between opsonophagocytic index (OI) and serum dilution, along with acceptable repeatability (CV ≤ 30%) and intermediate precision (CV ≤ 50%). Both preclinical and clinical serum samples exhibited potent bactericidal activity against diverse PspA families, independent of capsule type. Conclusions: This study provided a standardized framework to support the development and regulatory assessment of protein-based pneumococcal vaccines.