Introducing new recombinant protein antigens to existing pediatric combination vaccines is important in improving coverage and affordability, especially in low- and middle-income countries (LMICs). This case-study highlights the analytical and formulation challenges encountered with three recombinant non-replicating rotavirus vaccine (NRRV) antigens (t-NRRV formulated with Alhydrogel® adjuvant, AH) combined with a mock multidose formulation of a pediatric pentavalent vaccine used in LMICs. This complex formulation contained (1) vaccine antigens (i.e., whole-cell pertussis (wP), diphtheria (D), tetanus (T), Haemophilus influenza (Hib), and hepatitis B (HepB), (2) a mixture of aluminum-salt adjuvants (AH and Adju-Phos®, AP), and (3) a preservative (thimerosal, TH). Selective, stability-indicating competitive immunoassays were developed to monitor binding of specific mAbs to each antigen, except wP which required the setup of a mouse immunogenicity assay. Simple mixing led to the desorption of t-NRRV antigens from AH and increased degradation during storage. These deleterious effects were caused by specific antigens, AP, and TH. An AH-only pentavalent formulation mitigated t-NRRV antigen desorption; however, the Hib antigen displayed previously reported AH-induced instability. The same rank-ordering of t-NRRV antigen stability (P[8] > P[4] > P[6]) was observed in mock pentavalent formulations and with various preservatives. The lessons learned are discussed to enable future multidose, combination vaccine formulation development with new vaccine candidates.

For the batch release of vaccines, potency release assays are required. Non-animal in vitro tests have numerous advantages and are preferred; however, several vaccines are still released using in vivo assays. Their major drawback is the inherent variability with its practical implications. We quantified the variability of in vivo potency release assays for whole-cell pertussis, inactivated polio and meningococcal B (MenB) vaccines which showed large CV (Coefficient of Variation) ranging from 34% to 125%. As inherent variability might potentially be attributed to the highly variable immune system between individual animals, we evaluated the antibody titres to four MenB antigens in 344 individual outbred mice. These varied strongly, with more than 100-fold differences in antibody titres in responsive mice. Furthermore, within individual mice there was generally no correlation between the strengths of the responses to the four antigens. A mouse with a very low or no response to one antigen in many cases exhibited a strong response to another antigen. The large differences between individual animals is likely a considerable contributor to the inherent variability of in vivo potency assays. Our data again support the notion that it is preferred to move away from in vivo potency assays for monitoring batch to batch consistency as part of vaccine batch release testing.

Pertussis vaccines have drastically reduced the disease burden in humans since their implementation. Despite their success, pertussis remains an important global public health challenge. Bordetella pertussis resurgence could be a result of greater surveillance combined with improved diagnosis methods, changes in Bordetella pertussis biology, vaccine schedules, and/or coverage. Additionally, mechanisms of protection conferred by acellular pertussis (aP) and whole-cell pertussis (wP) vaccines differ qualitatively. There are no clear immune correlates of protection for pertussis vaccines. Pertussis antigens can induce toxin neutralizing antibodies, block adherence or engage complement mediated phagocytic/bactericidal killing.
We reviewed the existing evidence on antibody-mediated serum bactericidal and opsonophagocytic activity and discussed the relevance of these functional antibodies in the development of next-generation pertussis vaccines.
Current paradigm proposes that wP vaccines may confer greater herd protection than aP vaccines due to their enhanced clearance of bacteria from the nasopharynx in animal models. Functional antibodies may contribute to the reduction of nasal colonization, which differentiates aP and wP vaccines. Understanding the intrinsic differences in protective immune responses elicited by each class of vaccines will help to identify biomarkers that can be used as immunological end points in clinical trials.

UNASSIGNED: To describe the trend in the frequency of adverse events (AE) records associated to pertussis component vaccines between January 1st, 2015 and June 30th, 2020 in infants younger than 2-years-old in Chile, by reviewing the records submitted to the AEFI NIP, stratified by DTP-vaccine type, wP or aP.
UNASSIGNED: This was a retrospective observational study including all AEFI records of DTP (either aP or wP)-containing vaccines in the described sample. A descriptive analysis was performed according to vaccine type and AEFI, using MedDRA terminology.
UNASSIGNED: The total number of AEFI reports was 1,697: 815 corresponding to wP vaccines, 417 to aP vaccines, and 465 with unknown type. The reporting rates for the years 2015 to 2020 were 40.1, 56.2, 37.1, 24.7, 19.1, and 12.2 per 100,000 doses administered, respectively. The most reported AEFI were injection site erythema (42.9%), pyrexia (35.7%), and pain at the injection site (29.2%). Among all cases, 5.8% were SAEs (n = 98), 5.9% were SAEs for wP vaccines (n = 48) and 5.3% were for aP vaccines (n = 22).
UNASSIGNED: A significant decrease in AEFI reports was observed as of 2018, the year that the DTaP-IPV-HepB-Hib was introduced in the NIP.

Previous studies from low-resource countries have highlighted concerns surrounding non-specific effects of whole-cell pertussis vaccination, particularly in females. We sought to examine the effects of sex and birth weight on health services utilization following first exposure to whole-cell pertussis vaccine. Using a self-controlled case series design and by calculating relative incidence ratios (RIRs), we compared the relative incidence of emergency department visits and/or hospital admissions between sexes and between birth weight quintiles. Females had a higher relative incidence of events following vaccination compared to males (RIR = 1.13, 95% CI: 0.99, 1.30), which persisted after adjustment for birth weight (RIR = 1.12, 95% CI: 0.97, 1.28). We also observed a trend of increasing relative incidence of events over decreasing quintiles of birth weight; infants in the lowest quintile had a 26% higher relative event rate compared to the highest quintile, which was robust to adjustment for sex (Unadjusted RIR = 1.26, 95% CI: 1.01, 1.56; Adjusted RIR = 1.23, 95% CI: 0.99, 1.53). The risk of all-cause health services utilization immediately following vaccination, was elevated in female infants and infants having lower birth weight. Further study is warranted to determine if vaccine dosing should take infant weight into account.

Two types of vaccines are currently licensed for use against pertussis: whole-cell (wP) and acellular pertussis (aP). There is evidence that wP confers more durable immunity than aP, however wP has been more frequently associated with adverse events following immunisation (AEFI). A comparison of the frequency of AEFI with the first doses of wP and aP has not yet been clearly documented. This must be done in light of recent considerations to move towards a wP prime-aP boost vaccination strategy in low and middle-income countries.
To compare the frequency of AEFI associated with the first dose of the wP and aP vaccines. We also compared the frequency of AEFI associated with subsequent doses of wP.
This systematic review was carried out in strict accordance with the published protocol.
High heterogeneity amongst included one-armed studies did not allow for pooling of prevalence estimates. The prevalence estimates of AEFI at first vaccine dose of wP ranged from 0 to 75%, while the prevalence estimates of AEFI at first vaccine dose of aP ranges from 0 to 39%. The prevalence estimates of adverse events following second and third vaccine dose of wP ranged from 0 to 71% and 0 to 61%, respectively. Risk ratios among two-armed studies showed an increased risk of adverse events with first dose of wP compared to aP [local reaction RR 2.73 (2.33, 3.21), injection site pain RR 4.15 (3.24, 5.31), injection site swelling RR 4.38 (2.70, 7.12), fever over 38 °C RR 9.21 (5.39, 15.76), drowsiness RR 1.34 (1.18, 1.52) and vomiting RR 1.28 (0.91, 1.79)].
Our results confirm that, when comparing the first dose, wP is more reacotgenic than aP. The proposed wP prime followed by aP boost pertussis vaccine strategy should be approached with caution.

Physicochemical and immunochemical assays were applied to substantiate the relation between upstream processing and the quality of whole-cell pertussis vaccines. Bordetella pertussis bacteria were cultured on a chemically defined medium using a continuous cultivation process in stirred tank reactors to obtain uniform protein expression. Continuous culture favors the consistent production of proteins known as virulence factors. Magnesium sulfate was added during the steady state of the culture in order to diminish the expression of virulence proteins. Changes in gene expression and antigen composition were measured by microarrays, mass spectrometry and ELISA. Transcriptome and proteome data revealed high similarity between the biological triplicates demonstrating consistent cultivation of B. pertussis. The addition of magnesium sulfate resulted in an instant downregulation of the virulence genes in B. pertussis, but a gradual decrease of virulence proteins. The quantity of virulence proteins concurred highly with the potency of the corresponding whole-cell pertussis vaccines, which were determined by the Kendrick test. In conclusion, proteome analysis provided detailed information on the composition and proportion of virulence proteins present in the whole-cell preparations of B. pertussis. Moreover, proteome analysis is a valuable method to monitor the production process of whole-cell biomass and predict the product quality of whole-cell pertussis vaccines.

Pentavalent combination vaccines are important tools to strengthen the immunization programs in numerous countries throughout the world. A large number of countries have recognized the value of combination vaccines and have introduced whole cell pentavalent vaccines into their immunization programs. A phase III, multi-center, randomized, single blinded study of a fully liquid pentavalent DTwP-HepB-Hib investigational vaccine (Shan5™) was conducted across India in 2 cohorts: 15 toddlers were evaluated for safety and immunogenicity following a single booster dose (Cohort 1) followed by 1085 infants (Cohort 2) evaluated for immunogenicity and safety following 3-dose primary immunization of the investigational vaccine or a locally licensed comparator vaccine (Pentavac SD). Immune consistency analysis among 3 lots of the investigational vaccine, and immune non-inferiority analysis of pooled (3 lots) data of investigational vaccine vs. comparator vaccine were carried out in cohort 2. The vaccines demonstrated comparable safety and immune responses in cohort 1. In cohort 2, equivalent immune consistency among 3 lots was observed for all antigens except whole cell pertussis antigens, where a marginal variation was observed which was linked to the low power of the test and concluded to not have any clinical significance. Immune non-inferiority against the comparator vaccine was demonstrated for all 5 antigens. Safety results were comparable between vaccine groups. This investigational, fully-liquid, whole-cell pertussis (wP) containing new pentavalent vaccine was found to be safe and immunologically non-inferior to the licensed comparator vaccine.

To better understand vaccine-induced protection and its potential failure in light of recent whooping cough resurgence, we evaluated quantity as well as quality of memory T cell responses in B. pertussis-vaccinated preadolescent children. Using a technique based on flow cytometry to detect proliferation, cytokine production and phenotype of antigen-specific cells, we evaluated residual T cell memory in a cohort of preadolescents who received a whole-cell pertussis (wP; n=11) or an acellular pertussis vaccine (aP; n=13) during infancy, and with a median of 4 years elapsed from the last pertussis booster vaccine, which was aP for all children. We demonstrated that B. pertussis-specific memory T cells are detectable in the majority of preadolescent children several years after vaccination. CD4(+) and CD8(+) T cell proliferation in response to pertussis toxin and/or filamentous hemagglutinin was detected in 79% and 60% of the children respectively, and interferon-γ or tumor necrosis factor-α producing CD4(+) T cells were detected in 65% and 53% of the children respectively. Phenotyping of the responding cells showed that the majority of antigen-specific cells, whether defined by proliferation or cytokine production, were CD45RA(-)CCR7(-) effector memory T cells. Although the time since the last booster vaccine was significantly longer for wP-compared to aP-vaccinated children, their proliferation capacity in response to antigenic stimulation was comparable, and more children had a detectable cytokine response after wP- compared to aP-vaccination. This study supports at the immunological level recent epidemiological studies indicating that infant vaccination with wP induces longer lasting immunity than vaccination with aP-vaccines.