This study evaluated the formulation and stability of a quadrivalent glycoconjugate Shigella vaccine candidate based on four predominant strains (S. flexneri; 2a, 3a, and 6, and S. sonnei) covering ~64% of global Shigella infections. Each glycoconjugate antigen consists of a strain-specific O-polysaccharide (O-PS) covalently linked to the carrier protein IpaB, a component of the Shigella type III secretion system. First, selective competitive ELISAs were developed to measure antigenicity of the four O-PS-IpaB conjugates formulated with different adjuvants (i.e., Alhydrogel®, AH; Adju-phos®, AP; and CpG-1018®, CpG). Next, the monovalent S. sonnei O-PS-IpaB conjugate was studied to elucidate interactions with aluminum salt adjuvants (AH, AP) under different solution conditions. Third, the stability profiles of AH- or AP-adjuvanted S. sonnei O-PS-IpaB conjugate in various formulations (±CpG) were determined at different temperatures. Interestingly, incubation at 25 °C for 2 weeks resulted in increased antigenicity values when the antigen was bound to AP or AH, suggesting increased epitope exposure upon adjuvant binding. When bound to AP adjuvant at pH 5.8, the best glycoconjugate antigen stability was observed at elevated temperatures. The CpG adjuvant under these conditions, however, displayed incompatibility (i.e., material loss), presumably from precipitation due to lack of interaction with AP and presence of the detergent LDAO from the bulk antigen buffer. In contrast, the glycoconjugate antigen and CpG adjuvant were both bound to the AH adjuvant and stable at 2–8 °C, pH 7.0. This AH-CpG formulation of the O-PS-IpaB conjugate antigens was identified as a promising candidate for future animal immunogenicity testing.

Inactivated, whole-cell pertussis (wP) vaccines remain at the frontline in the global fight against the resurgence of whooping cough, especially in low- and middle-income countries. However, the reliance on the intracerebral mouse potency test (ic-MPT or Kendrick assay) as the standard batch release assay is extremely burdensome for commercial wP vaccine production. The ic-MPT is technically challenging, labor intensive, and incongruous with modern animal welfare guidelines. Replacing the ic-MPT with a whole-cell Bordetella pertussis enzyme-linked immunosorbent assay, the so-called pertussis serology potency test, has shown promise but has been difficult to implement in practice. In this report, we tested the hypothesis that potent and subpotent wP vaccines have distinct serological profiles in mice that could be developed as a substitute for the ic-MPT. We established an accelerated decay (thermal stress) protocol in which wP, in the context of diphtheria-tetanus-whole-cell pertussis, was rendered >10-fold less effective than unstressed vaccine when evaluated in a mouse model of B. pertussis lung clearance following intranasal challenge. We then screened immune sera on a limited B. pertussis Tahoma I proteome array and identified >30 antigens whose antibody reactivity profiles either increased, decreased, or were unchanged as a function of wP potency. Moreover, virtually all the "indicator" antigens identified are known virulence factors or reactive with human convalescent sera, thereby establishing a potential link between wP potency and pertussis infection and immunity. These results support the development of a limited B. pertussis antigen array as a stability-indicating surrogate potency assay for the ic-MPT. IMPORTANCE:Whooping cough (pertussis) is a highly contagious respiratory disease caused by the Gram-negative bacterium, Bordetella pertussis. Globally, tens of millions of whole-cell pertussis (wP) vaccines are administered annually. Whole-cell pertussis vaccines are logistically complex to manufacture and get to market because of the need for each batch of vaccine to be evaluated in a highly laborious and challenging potency test known as the Kendrick assay, which involves mouse intracerebral challenges with B. pertussis. In this report, we describe efforts to develop a serology-based substitute for the Kendrick assay that relies on profiling antibody responses to wP vaccines.

Disease X in the Tropics, preventing the next pandemic: how to accelerate spillover prevention and vaccine preparedness?

Traditional vaccination, based on the core theory proposed by Louis Pasteur, has been instrumental in increasing life expectancy and eradicating diseases. However, global vaccine access is limited due to high production costs, limited efficacy over time, safety concerns, economic inequality, and hesitancy. Traditional approaches for vaccine manufacturing, in general, are time consuming, and commercial production of subunit vaccines faces technical challenges and involves expensive antigen purification procedures. Furthermore, the emergence of new diseases due to mutations and environmental exposures requires innovative approaches to overcome these challenges and address persistent health risks posed by vaccine-preventable diseases. Vaccines along with therapeutic interventions have proven to be effective against various infectious diseases and cancer, and recent advancements in omics sciences and bioinformatics tools have further accelerated vaccine research and development. Omics technologies encompass various "-omic" fields, such as genomics, transcriptomics, proteomics, metabolomics, and immunomics, enabling comprehensive analysis of cellular molecules and identification of potential vaccine antigens. Omics approaches have revolutionized vaccine design, particularly "reverse vaccinology," which involves genome-based identification of vaccine antigens. Proteogenomics, combining proteomics and genomics, facilitates the discovery of new peptides for vaccine development. Immunopeptidomics, employing mass spectrometry to identify MHC-bound peptides, plays a crucial role in finding vaccine targets and developing effective vaccines by identifying T-cell epitopes. These advancements provide a powerful foundation for the development of reliable and targeted vaccines.

In this work, we describe compatibility assessments of a recombinant, trivalent non-replicating rotavirus vaccine (t-NRRV) candidate with a mock trivalent Sabin inactivated polio vaccine (t-sIPV). Both t-sIPV and t-NRRV are incompatible with thimerosal (TH), a preservative commonly used in pediatric pentavalent combination vaccines (DTwP-Hib-HepB) distributed in low- and middle-income countries (LMICs), preventing the development of a heptavalent combination. The compatibility of t-NRRV with a mock DTwP-Hib-HepB formulation is described in a companion paper. This case study highlights the analytical and formulation challenges encountered when combining a mock t-sIPV vaccine (unadjuvanted) with Alhydrogel® (AH) adjuvanted t-NRRV. Selective and stability-indicating competition ELISAs were implemented to monitor antibody binding to each of the six antigens (±AH). Simple mixing caused the undesired desorption of t-NRRV from AH with the concomitant binding of t-sIPV to AH. Although the former effect was mitigated by dialyzing sIPV bulks, decreased sIPV storage stability was observed at accelerated temperatures in the bivalent combination with a rank-ordering of P[8] > P[6] > P[4] and sIPV3 > sIPV2 > sIPV1. The compatibility of AH-adsorbed t-sIPV with alternative preservatives was evaluated, and parabens (methyl, propyl) were identified for potential use in this multi-dose bivalent formulation. Along with a companion paper, the lessons learned are discussed to facilitate the future formulation development of pediatric combination vaccines with new antigens.

Inactivated, whole cell pertussis (wP) vaccines remain at the frontline in the global fight against the resurgence of whooping cough, especially in low- and middle-income countries. However, the reliance on the intracerebral mouse potency test (ic-MPT or Kendrick assay) as the standard batch release assay is challenging for the production of commercial wP vaccines. The ic-MPT is technically challenging, labor intensive and, arguably, incongruous with modern animal welfare guidelines. Replacing the ic-MPT with a whole cell Bordetella pertussis ELISA, the so-called pertussis serology potency test (PSPT), has shown promise, but has been difficult to implement in practice. In this report, we tested the hypothesis that potent and subpotent wP vaccines have distinct serologic profiles in mice that could be developed as a substitute for the ic-MPT. We first established an accelerated decay (thermal stress) protocol in which wP, in the context of DTwP, was rendered >10-fold less effective than unstressed vaccine when evaluated in a mouse model of B. pertussis lung clearance following intranasal challenge. We then screened immune sera on a limited B. pertussis Tahoma I proteome array and identified >30 antigens whose antibody reactivity profiles increased, decreased or were unchanged as a function of wP potency. Interestingly, virtually all the indicator antigens identified are known virulence factors or reactive with human convalescent sera, thereby establishing a potential link between wP potency and pertussis infection and immunity. These results bode well for the development of a limited B. pertussis antigen array as a stability-indicating surrogate potency assay for the ic-MPT. ### Competing Interest Statement The authors have declared no competing interest.

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.

Drug and vaccine delivery have received considerable attention in recent years [...].

Recombinant adeno-associated viruses (rAAVs) are a preferred vector system in clinical gene transfer. A fundamental challenge to formulate and deliver rAAVs as stable and efficacious vaccines is to elucidate interrelationships between the vector’s physicochemical properties and biological potency. To this end, we evaluated an rAAV-based COVID-19 vaccine candidate which encodes the Spike antigen (AC3) and is produced by an industrially-compatible process. First, state-of-the-art analytical techniques were employed to determine key structural attributes of AC3 including primary and higher-order structures, particle size, empty/full capsid ratios, aggregates and multi-step thermal degradation pathway analysis. Next, several quantitative potency measures for AC3 were implemented and data were correlated with the physicochemical analyses on thermal-stressed and control samples. Results demonstrate links between decreasing AC3 physical stability profiles, in vitro transduction efficiency in a cell-based assay, and importantly, in vivo immunogenicity in a mouse model. These findings are discussed in the general context of future development of rAAV-based vaccines candidates as well as specifically for the rAAV vaccine application under study.

The development of multi-dose, subunit vaccine formulations can be challenging since antimicrobial preservatives (APs) often destabilize protein antigens. In this work, we evaluated Human Papillomavirus (HPV) Virus-Like Particles (VLPs) to determine if combining different APs used in approved parenteral products, each at lower concentrations than used alone, would maintain both antimicrobial effectiveness and antigen stability. To identify promising AP combinations, two different screening strategies were utilized: (1) empirical one-factor-at-a-time (OFAT) and (2) statistical design-of-experiments (DOE). Seven different APs were employed to screen for two- and three-AP combinations using high-throughput methods for antimicrobial effectiveness (i.e., microbial growth inhibition assay and a modified European Pharmacopeia method) and antigen stability (i.e., serotype-specific mAb binding to conformational epitopes of HPV6, 11, 16 VLPs by ELISA). The OFAT and DOE approaches were complementary, such that initial OFAT results (and associated lessons learned) were subsequently employed to optimize the combinations using DOE. Additional validation experiments confirmed the final selection of top AP-combinations predicted by DOE modeling. Overall, 20 candidate multi-dose formulations containing two- or three-AP combinations were down-selected. As described in Part 2 (companion paper), long-term storage stability profiles of aluminum-adjuvanted, quadrivalent HPV VLP formulations containing these lead candidate AP combinations are compared to single APs.

Implementation of inactivated polio vaccines (IPV) containing Sabin strains (sIPV) will further enable global polio eradication efforts by improving vaccine safety during use and containment during manufacturing. Moreover, sIPV-containing vaccines will lower costs and expand production capacity to facilitate more widespread use in low- and middle-income countries (LMICs). This review focuses on the role of vaccine formulation in these efforts including traditional Salk IPV vaccines and new sIPV-containing dosage forms. The physicochemical properties and stability profiles of poliovirus antigens are described. Formulation approaches to lower costs include developing multidose and combination vaccine formats as well as improving storage stability. Formulation strategies for dose-sparing and enhanced mucosal immunity include employing adjuvants (e.g. aluminum-salt and newer adjuvants) and/or novel delivery systems (e.g. ID administration with microneedle patches). The potential for applying these low-cost formulation development strategies to other vaccines to further improve vaccine access and coverage in LMICs is also discussed.

BioanalysisAhead of Print EditorialAnalytical considerations to support expeditious formulation development of vaccinesPrashant Kumar & Vibhuti AgrahariPrashant Kumar *Author for correspondence: E-mail Address: prashant.kumar@ku.eduhttps://orcid.org/0000-0002-4959-3156Department of Pharmaceutical Chemistry, Vaccine Analytics & Formulation Center, University of Kansas, Lawrence, KS 66047, USA & Vibhuti Agrahari **Author for correspondence: E-mail Address: vibhuti-agrahari@ouhsc.eduhttps://orcid.org/0000-0003-1884-1644Department of Pharmaceutical Sciences, University of Oklahoma Health Sciences Center, 1110 N. Stonewall Avenue, Oklahoma City, OK 73117, USAPublished Online:11 Jul 2023https://doi.org/10.4155/bio-2023-0090AboutSectionsView ArticleView Full TextPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareShare onFacebookTwitterLinkedInRedditEmail View articleKeywords: analysiscorrelationhigh-throughputlive viral vaccineslow- and middle-income countriesmicrofluidicpotency assaysrotavirusRT-qPCRTCID50References1. Verch T, Trausch JJ, Shank-Retzlaff M. Principles of vaccine potency assays. Bioanalysis 10(3), 163–180 (2018).Link, CAS, Google Scholar2. Sanyal G. Development of functionally relevant potency assays for monovalent and multivalent vaccines delivered by evolving technologies. NPJ Vaccines 7(1), 1–10 (2022).Crossref, Medline, Google Scholar3. Schofield TL. Vaccine stability study design and analysis to support product licensure. Biologicals 37(6), 387–396 (2009).Crossref, Medline, CAS, Google Scholar4. Kumar P, Shukla RS, Patel A et al. Formulation development of a live attenuated human rotavirus (RV3-BB) vaccine candidate for use in low- and middle-income countries. Hum. Vaccin. Immunother. 17(7), 2298–2310 (2021).Crossref, Medline, CAS, Google Scholar5. Kumar P, Pullagurla SR, Patel A et al. Effect of formulation variables on the stability of a live, rotavirus (RV3-BB) vaccine candidate using in vitro gastric digestion models to mimic oral delivery. J. Pharm. Sci. 110(2), 760–770 (2021).Crossref, Medline, CAS, Google Scholar6. Wan Y, Gupta V, Bird C et al. Formulation development and improved stability of a combination measles and Rubella live-viral vaccine dried for use in the Nanopatch™ microneedle delivery system. Hum. Vaccin. Immunother. 17(8), 2501–2516 (2021).Crossref, Medline, CAS, Google Scholar7. Kumar P, Wang M, Kumru OS et al. Correlating physicochemical and biological properties to define critical quality attributes of a recombinant AAV vaccine candidate. Mol. Ther. - Methods Clin. Dev. 30, 103–121 (2023).Crossref, CAS, Google Scholar8. InDevR. VaxArray®platform. www.indevr.com/products/vaxarray/Google Scholar9. LumaCyte. Vaccines & viral infectivity. www.lumacyte.com/application/vaccines-viral-infectivityGoogle Scholar10. Bio-Rad. qPCR assay design and optimization. www.bio-rad.com/en-us/applications-technologies/qpcr-assay-design-optimization?ID=LUSO7RIVKGoogle Scholar11. Cumming GF, Fidler F, Vaux DL. Error bars in experimental biology. J. Cell Biol. 177(1), 7–11 (2007).Crossref, Medline, CAS, Google Scholar12. Pullagurla SR, Kumar P, Ogun O et al. Modeling the long-term 2–8 degrees C stability profiles of a live, rotavirus vaccine candidate (RV3-BB) in various liquid formulations via extrapolations of real-time and accelerated stability data. Biologicals 75, 21–28 (2022).Crossref, Medline, CAS, Google ScholarFiguresReferencesRelatedDetails Ahead of Print STAY CONNECTED Metrics Downloaded 0 times History Received 11 May 2023 Accepted 27 June 2023 Published online 11 July 2023 Information© 2023 Newlands PressKeywordsanalysiscorrelationhigh-throughputlive viral vaccineslow- and middle-income countriesmicrofluidicpotency assaysrotavirusRT-qPCRTCID50Financial & competing interests disclosureThe authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.No writing assistance was utilized in the production of this manuscript.PDF download

Rotavirus infections remain a leading cause of morbidity and mortality among infants residing in low- and middle-income countries. To address the large need for protection from this vaccine-preventable disease we are developing a trivalent subunit rotavirus vaccine which is currently being evaluated in a multinational Phase 3 clinical trial for prevention of serious rotavirus gastroenteritis. Currently, there are no universally accepted in vivo or in vitro models that allow for correlation of field efficacy to an immune response against serious rotavirus gastroenteritis. As a new generation of non-replicating rotavirus vaccines are developed the lack of an established model for evaluating vaccine efficacy becomes a critical issue related to how vaccine potency and stability can be assessed. Our previous publication described the development of an in vitro ELISA to quantify individual vaccine antigens adsorbed to an aluminum hydroxide adjuvant to address the gap in vaccine potency methods for this non-replicating rotavirus vaccine candidate. In the present study, we report on concordance between ELISA readouts and in vivo immunogenicity in a guinea pig model as it relates to vaccine dosing levels and sensitivity to thermal stress. We found correlation between in vitro ELISA values and neutralizing antibody responses engendered after animal immunization. Furthermore, this in vitro assay could be used to demonstrate the effect of thermal stress on vaccine potency, and such results could be correlated with physicochemical analysis of the recombinant protein antigens. This work demonstrates the suitability of the in vitro ELISA to measure vaccine potency and the correlation of these measurements to an immunologic outcome.

The human body poses a spectrum of biological mechanisms operating at different levels that are important for its normal functioning and development [...].

Formulation development was performed with the live, attenuated, human neonatal rotavirus vaccine candidate (RV3-BB) with three main objectives to facilitate use in low- and middle- income countries including (1) a liquid, 2–8°C stable vaccine, (2) no necessity for pre-neutralization of gastric acid prior to oral administration of a small-volume dose, and (3) a low-cost vaccine dosage form. Implementation of a high-throughput RT-qPCR viral infectivity assay for RV3-BB, which correlated well with traditional FFA assays in terms of monitoring RV3-BB stability profiles, enabled more rapid and comprehensive formulation development studies. A wide variety of different classes and types of pharmaceutical excipients were screened for their ability to stabilize RV3-BB during exposure to elevated temperatures, freeze-thaw and agitation stresses. Sucrose (50–60% w/v), PEG-3350, and a solution pH of 7.8 were selected as promising stabilizers. Using a combination of an in vitro gastric digestion model (to mimic oral delivery conditions) and accelerated storage stability studies, several buffering agents (e.g., succinate, adipate and acetate at ~200 to 400 mM) were shown to protect RV3-BB under acidic conditions, and at the same time, minimize virus destabilization during storage. Several optimized RV3-BB candidate formulations were identified based on negligible viral infectivity losses during storage at 2–8°C and −20°C for up to 12 months, as well as by relative stability comparisons at 15°C and 25°C (up to 12 and 3 months, respectively). These RV3-BB stability results are discussed in the context of stability profiles of other rotavirus serotypes as well as future RV3-BB formulation development activities.

Despite solid evidence of the success of rotavirus vaccines in saving children from fatal gastroenteritis, more than 82 million infants worldwide still lack access to a rotavirus vaccine. The main barriers to global rotavirus vaccine coverage include cost, manufacturing capacity and suboptimal efficacy in low- and lower-middle income countries. One vaccine candidate with the potential to address the latter is based on the novel, naturally attenuated RV3 strain of rotavirus, RV3-BB vaccine administered in a birth dose strategy had a vaccine efficacy against severe rotavirus gastroenteritis of 94% at 12 months of age in infants in Indonesia. To further develop this vaccine candidate, a well-documented and low-cost manufacturing process is required. A target fully loaded cost of goods (COGs) of <=$3.50 per course of three doses was set based on predicted market requirements. COGs modelling was leveraged to develop a process using Vero cells in cell factories reaching high titers, reducing or replacing expensive reagents and shortening process time to maximise output. Stable candidate liquid formulations were developed allowing two-year storage at 2-8 degrees C. In addition, the formulation potentially renders needless the pretreatment of vaccinees with antacid to ensure adequate gastric acid neutralization for routine oral vaccination. As a result, the formulation allows small volume dosing and reduction of supply chain costs. A dose ranging study is currently underway in Malawi that will inform the final clinical dose required. At a clinical dose of <= 6.3 log(10) FFU, the COGs target of <=$3.50 per three dose course was met. At a clinical dose of 6.5 log(10) FFU, the final manufacturing process resulted in a COGs that is substantially lower than the current average market price, 2.44 USD per dose. The manufacturing and formulation processes were transferred to BioFarma in Indonesia to enable future RV3-BB vaccine production. (C) 2021 Batavia Biosciences BV. Published by Elsevier Ltd.

During the formulation and development of therapeutic proteins, a thorough understanding of their structure and stability is essential. In this chapter, we discuss the most common methods used for this purpose and their individual advantages and disadvantages. This discussion is organized in terms of levels of structure (primary, secondary, tertiary, and quaternary including aggregation). Special attention is given to peptide mapping, circular dichroism, infrared and Fourier transform methods, ultraviolet absorbance, intrinsic and extrinsic fluorescence, and dynamic and static light scattering. Brief discussions concerning micro-flow imaging, nanoparticle tracking analysis, analytical ultracentrifugation, micro-calorimetry and hydrogen/deuterium exchange, and viscosity measurements are also included. Detailed examples are illustrated in figures and their legends.

In this work, two different in vitro gastric digestion models were used to evaluate the stability of a live attenuated rotavirus vaccine candidate (RV3-BB) under conditions designed to mimic oral delivery in infants. First, a forced-degradation model was established at low pH to assess the buffering capacity of formulation excipients and to screen for RV3-BB stabilizers. Second, a sequential-addition model was implemented to examine RV3-BB stability under conditions more representative of oral administration to infants. RV3-BB rapidly inactivated at < pH 5.0 (37 degrees C, 1 h) as measured by an infectivity RT-qPCR assay. Pre-neutralization with varying volumes of infant formula (Enfamil (R)) or antacid (Mylanta (R)) conferred partial to full protection of RV3-BB. Excipients with sufficient buffering capacity to minimize acidic pH inactivation of RV3-BB were identified (e.g., succinate, acetate, adipate), however, they concomitantly destabilized RV3-BB in accelerated storage stability studies. Both effects were concentration dependent, thus excipient optimization was required to design candidate RV3-BB formulations which minimize acid-induced viral inactivation during oral delivery while not destabilizing the vaccine during long-term 2-8 degrees C storage. Finally, a statistical Design-of-Experiments (DOE) study examining RV3-BB stability in the in vitro sequential-addition model identified key formulation parameters likely affecting RV3-BB stability during in vivo oral delivery.