Development strategy and lessons learned for a 10-valent pneumococcal conjugate vaccine (PNEUMOSIL®)

ABSTRACT Pneumococcal conjugate vaccines (PCVs) have proven to be the best way to prevent severe childhood pneumococcal disease but until recently have been difficult for many countries to afford sustainably. In 2008, the Serum Institute of India, Pvt. Ltd. and PATH entered into a collaboration, funded in part by the Bill & Melinda Gates Foundation, to respond to this problem by developing a PCV designed to be affordable, accessible, and protective against the pneumococcal serotypes causing the most morbidity and mortality in low- and middle-income countries. The resulting 10-valent PCV (PNEUMOSIL®) received World Health Organization prequalification in December 2019 – making it just the third PCV to be certified as an option for Gavi, the Vaccine Alliance-eligible countries – and is being made available at a Gavi price of US$2/dose. The task of developing a state-of-the-art, yet lower-priced, PCV required public-private collaboration across geographies and yielded a variety of successes and learnings useful to the vaccine development field. Key among the learnings were factors related to manufacturing strategy and optimization, serotype selection, flexibility, early risk detection and mitigation, partner trust and continuity across similar-class products, complementary business philosophies, and early clarity of purpose.


Introduction
Pneumonia remains the top infectious disease killer of children under five worldwide, with most mortality in low-and middleincome countries (LMICs). 1 Pneumococcal conjugate vaccines (PCVs) have helped reduce severe childhood pneumonia and other pneumococcal diseases like meningitis, sepsis, and otitis media since first licensure in 2000. 2,3 Relatively high PCV prices have, however, made access cost-prohibitive for many countries and only possible for low-income economies eligible for Gavi, the Vaccine Alliance financial support. 4 Additionally, PCV financing disproportionately appropriates donor funds compared to other vaccines -approximately 43% of Gavi's 2016-2020 vaccine expenditures and over 24% anticipated through 2025. 5,6 PCVs are extremely complex to manufacture and, until 2019, only two multinational corporations had achieved World Health Organization (WHO) prequalification. This duopoly has limited supply and stifled competitive market forces that drive prices down. To assuage these dynamics, Gavi created the pneumococcal Advance Market Commitment (AMC) -a financing mechanism designed to incentivize PCV manufacturers to scale up supply and encourage additional manufacturers to develop PCVs suitable for Gavi-supported countries. Still, price and supply barriers leave over 55 million children without access to PCVs (Figure 1) 7 -underscoring an urgency for more affordable vaccines that protect on par with existing ones, alleviate financial burdens on countries and donors, and foster sustainable, universal PCV access.
In 2008, Serum Institute of India, Pvt. Ltd. (SIIPL) and PATH initiated the development of such a PCV, with funding partly provided by the Bill & Melinda Gates Foundation. A decade later, the resulting 10-valent PCV -PNEUMOSIL® -is WHOprequalified, AMC-approved, and licensed in India. 8 PNEUMOSIL's LMIC price is an unprecedented US$2 per dose -a reduction of over 30% from the Gavi-supported price for other PCVs and dramatically less than non-Gavi prices. 9 It is also the first PCV designed specifically to target serotypes most likely to cause invasive pneumococcal disease (IPD) in Africa and Asia (the highest-burden regions), and Latin America and the Caribbean (LAC). 10 Herein, we discuss factors contributing to PNEUMOSIL's successful development, learnings, and future considerations. The purpose is to inform others seeking to accelerate the development, lower the costs, and preserve the quality of vaccines for high-burden, resource-limited populations.

Collaboration structure and strategy
In 2006, PATH received a grant from the Bill & Melinda Gates Foundation for its pneumococcal vaccine project (PVP) to accelerate the development of affordable and accessible pneumococcal vaccines for LMICs. Initially, PVP's target was protein vaccines but delays in development led to higher demand for a lower-cost PCV -shifting PVP's top priority to affordable PCVs.
The 2008 SIIPL-PATH collaboration built upon an existing collaboration evaluating novel technologies for conjugate vaccines. The partners decided to focus on validated technologies to minimize risk and timelines to licensure. They also applied learnings from a previous partnership with WHO and African countries that developed and introduced MenAfriVac® -a lowpriced Neisseria meningitidis serogroup A conjugate vaccine that eliminated meningitis A epidemics in Africa's meningitis belt. 11 PNEUMOSIL's developmentgoal was two-fold: WHO prequalification to catalyze improved LMIC PCV access and Indian marketing authorization (MA). In a two-staged global access strategy, initial collaboration agreements adhered to broad provisions that ensured appropriate pricing and distribution, with terms specifying general affordability and supply. Later, long-term, large-scale clinical development resourcing (from SIIPL in India and the Bill & Melinda Gates Foundation in Africa) afforded sufficient confidence in program continuity for more concrete global access terms to be established using tiered pricing, including a target price of 2 USD per dose for LMICs, contingent upon demand.

Serotype selection
The challenge was to develop a more affordable PCV with comparable performance, quality, and serotype coverage in LMICs as existing vaccines. Pneumococcal Global Serotype Project and other disease burden data helped prioritize serotypes causing the highest pediatric IPD incidence in Africa, Asia, and LAC. Ultimately, ten serotypes (1, 5, 6A, 6B, 7F, 9V, 14, 19A, 19F, 23F) were chosen for optimal balance of cost, coverage, and competitiveness with existing PCVs. 10 Accordingly, PNEUMOSIL's theoretical cumulative coverage (71%) in these regions is comparable to the other WHOprequalified 10-and 13-valent PCVs (66% [Synflorix™] and 76% [Prevnar 13®], respectively). 10 Serotypes 6A and 19A were emerging as important causes of IPD in target regions, so were critical inclusions to ensure that PNEUMOSIL matched or exceeded other PCVs for serotype coverage.

Formulation, presentation, and manufacturing optimization
Decisions around dose level, carrier protein, conjugation technology, adjuvant (at the lowest possible concentration to minimize reactogenicity), and preservative (for multi-dose formulation) were all based upon proven approaches to minimize risk. Additional strategic input from PVP's consultants and scientific advisory board (SAB) was also considered. Vaccine specifications also followed WHO's TRS 977 Annex 3 guidance and the AMC target product profile (TPP). Importantly, SIIPL optimized three innovative manufacturing processes that substantially lowered costs while maintaining high vaccine quality -carrier protein production, polysaccharide production, and conjugation efficiency. A key innovation was early implementation and optimization of an efficient manufacturing process for the CRM 197 carrier protein using Pfenex, Inc.'s recombinant expression technology. Another innovation involved optimizing an efficient conjugation process using CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate) chemistry in collaboration with Fina Biosolutions.

Preclinical development
PNEUMOSIL underwent seven toxicology studies in India in rats and rabbits with regulatory oversight by the Review Committee on Genetic Manipulation (RCGM) of India's Department of Biotechnology. Single-and repeat-doses (even at >20 times the human dose) were well tolerated -findings similar to those with a licensed comparator PCV. Serotypespecific immune response assessment used an enzyme-linked immunosorbent assay (ELISA) to quantitate serotype-specific immunoglobulin G (IgG) and a multiplexed opsonophagocytic activity (MOPA) assay to estimate functional antibodies. Total IgG and functional antibody responses were equivalent to those of the licensed PCV comparator across all vaccine serotypes. Similar analyses with multiple lots after 1 year of manufacture showed comparable IgG and OPA antibody titers to those at the time of manufacture. Based on these results, RCGM approved the Indian national regulatory authority, the Drug Controller General of India (DCGI), to advance PNEUMOSIL into human studies.

An evolving clinical strategy
PNEUMOSIL's clinical development plan evolved as circumstances changed (Table 1). Originally, SIIPL and PATH planned to partner on the trials required for Indian MA, which would constitute the basis for WHO prequalification. When it became clear that the Phase 1 trial in India was restricted to a safety study in adults from a regulatory perspective, the team adjusted strategy to conduct a parallel Phase 1/2 clinical trial in Africa to generate early safety and immunogenicity data in adults, toddlers, and infants (the target population). The team determined that the quickest way to WHO prequalification and Indian MA was to undertake both India and Africa clinical development in parallel. Doing so would de-risk the program from potential regionspecific delays and generate clinical data from diverse populations. In the resulting separate but complementary programs, SIIPL led and funded the Indian studies for MA, and PATH (with a grant from the Bill & Melinda Gates Foundation) sponsored the African studies required for WHO prequalification. To maintain cohesion, the SIIPL and PATH teams drove the evolving clinical strategy across regulatory landscapes and technical consensusbuilding between the Indian national regulatory authority, key health agencies, and WHO. The strategy shift meant that WHO prequalification would now be based on a Notice of Compliance (NOC) for export from DCGI -an approach informed by consultations with Indian regulators and WHO.
The revised strategy's advantages included preserving the pathway to Indian MA and introduction, minimizing time to  Control Organization guidelines and one of two WHOapproved schedules). It met all primary and secondary objectives (Table 2), with PNEUMOSIL demonstrating acceptable safety and tolerability and eliciting immune responses comparable to Prevnar 13 and Synflorix for all 10 serotypes.

African clinical studies (2015-2020).
After considering various locations, a site run by MRC Unit in The Gambia -part of the London School of Hygiene and Tropical Medicine (LSHTM) -was selected. Factors favoring The Gambia included previous PATH/SIIPL experience with the MRC site, the country's expertise in pneumococcal disease and vaccines, and large efficacy study experience with a PCV. 15 Studies included a Phase 1/2 age de-escalation study and a non-inferiority Phase 3 trial required for WHO prequalification. CRO FHI Clinical supported both trials. Pneumococcal serology occurred at UCL while other concomitant vaccine laboratory testing occurred at Public Health England, Q 2 Solutions, Cincinnati Children's Hospital and Medical Center, and the US Centers for Disease Control and Prevention.
The Phase 1/2 trial evaluated PNEUMOSIL, first, in 34 healthy PCV-naïve adults 18-40 years old, then in 112 PCV-primed children 12-15 months old, and finally in 200 PCV-naïve infants 6-8 weeks old using a 3 + 1 schedule. The comparator for infants and children was Prevnar 13. PNEUMOSIL demonstrated an acceptable safety and tolerability profile and was immunogenic for all 10 vaccine serotypes -supporting entering Phase 3. 12 The Gambian, multi-site Phase 3 enrolled 2,250 infants to receive either PNEUMOSIL or Synflorix in a primary 3 + 0 To assess the safety and tolerability of a 3 + 0 schedule of PNEUMOSIL, alone and in comparison to Prevnar 13.
Alone and in comparison to Prevnar 13: • PNEUMOSIL met all primary and secondary endpoints, including safety and tolerability, immunological noninferiority to Synflorix per WHO specifications (TRS 977 Annex 3) for all 10 vaccine serotypes, lot-to-lot consistency, and noninterference with routine childhood immunizations (Table 3). 13 Immune response persistence was demonstrated at 10 months of age following the 3-dose priming series in the Gambian Phase 1/2 study 12 and 12 months following the booster immunization in the Gambian Phase 3 study. 13 The African and Indian clinical programs achieved a complementary data package that provided a comprehensive picture of PNEUMOSIL's performance relative to both WHOprequalified PCVs. Using Synflorix as comparator in the Gambian Phase 3 added value because PNEUMOSIL had been evaluated in India and The Gambia with Prevnar 13 as comparator. Furthermore, Synflorix was highly efficacious against vaccine-type IPD, pneumonia, and otitis media in infants and children in randomized controlled trials 16,17 and effective against IPD and pneumonia in infants in sub-Saharan Africa. 18,19 (Regarding Prevnar 13 and Synflorix, a recent WHO position paper stated that "the currently available evidence does not point to a difference between the two vaccines in their net impact on the overall pneumococcal disease burden.") 20 Strengthening comprehensiveness, the Indian Phase 3 evaluated PNEUMOSIL head-to-head against Prevnar 13 and Synflorix (manuscript in preparation).

Approval milestones and looking forward
PNEUMOSIL received the Indian Export NOC in December 2018, after which SIIPL applied for WHO prequalification in January 2019 with support from PATH, FHI Clinical, and 4Clinics medical writers. PNEUMOSIL received WHO prequalification in December 2019, an AMC supply agreement in June 2020, and Indian MA in July 2020. 21, 22 As a result, PNEUMOSIL is now an option for LMICs to consider for PCV introduction or program switch. Efforts are underway to share information about this new vaccine with LMICs and to continue evaluating the vaccine to inform public health decisions. SIIPL will conduct a post-licensure nasopharyngeal carriage study to obtain data on vaccine effectiveness required by WHO as a condition of prequalification. Ongoing Table 3. Primary and secondary objectives and endpoints for Phase 3 clinical study in The Gambia to evaluate PNEUMOSIL® in a 3-dose primary series with booster (3 + 1) 13 Objective Objective description Endpoints Primary (immunogenicity) Demonstrate equivalent immune responses to the 10 vaccine serotypes across 3 different lots when measured 4 weeks post dose 3.

Lot consistency
• Serotype-specific IgG GMC measured 4 weeks post dose 3 Demonstrate non-inferior immune responses for at least 7 of the 10 vaccine serotypes in comparison to matched serotypes or lowest responder in Synflorix™.

Non-inferiority
• % subjects with serotype-specific IgG concentrations ≥ clinical studies are evaluating PNEUMOSIL in the other WHOrecommended schedule (2 + 1) -one by SIIPL in India to meet MA regulatory requirements and inform uptake and the other by PATH and MRC Unit The Gambia at LSHTM to augment data for LMIC decision-making. A 2 + 1 schedule could be beneficial because the third vaccination at 9 to 18 months of age provides a better boost to protective antibodies and potentially contributes to stronger herd protection. 20,23

Keys to success and lessons learned
To inform others seeking to accelerate vaccine development for LMICs, PNEUMOSIL development yielded the following keys to success and learnings: Manufacturing optimization to reduce cost of goods sold (COGS) and serotype selection are key for reducing PCV price. Since PCVs are among the most complex and expensive vaccines to manufacture, optimizing manufacturing processes is difficult and the business risks of changing processes can be disincentivizing. Developed from scratch, however, PNEUMOSIL was not beholden to these dynamics. Manufacturing optimization could proceed in a manner that improved the vaccine's quality/performance, reduced its COGS, and supported its high-volume/lowprice value proposition. The decision to include only serotypes most relevant for LMICs (especially 6A and 19A) while omitting those less applicable proved invaluable to PNEUMOSIL's advantageous balance of coverage, performance, and cost for LMICs. Since serotypes vary by region and each adds expense, risk, and time to development and manufacturing, PNEUMOSIL's strategy achieved the best of both worlds -coverage and immunity comparable to prequalified PCVs (if not superior in some cases) at a lower price point.
Regulatory management and clinical consensus-building helped detect and mitigate risks early. Taking a vaccine candidate into humans comes with risk; therefore, having safeguards and the necessary expertise to agilely resolve issues is important. A key to PNEUMOSIL's success was adjusting the clinical and regulatory strategy when roadblocks arose, which was possible because the skillsets were in place to recognize risks and mitigate them. SIIPL and PATH's cross-functional team of experts identified the regulatory landscape's evolution in India early (e.g., through SIIPL's engagement with DCGI and ministry agencies and multi-agency consensus meetings), which otherwise could have incurred debilitating delays for the program if undetected. The PNEUMOSIL program was significantly ahead of the PCV-specific regulatory and industry curve at the time and, therefore, needed intense negotiations to build consensus around the trans-continental program. The team worked together to consider scenarios and build consensus around regulatory options internally and, then, coordinated with regulators in India and WHO to identify an optimal alternative and lay a foundation to develop an appropriate clinical strategy.
Clinical team flexibility enabled a smooth transition to a dual pathway. The cross-geographical clinical teams' flexibility to react to and execute a new strategy enabled the parallel India-Gambia clinical plan to come to fruition. The willingness to change tack and adjust to fit circumstances saved valuable time and enabled PNEUMOSIL's market entry earlier than otherwise possible.
Challenges, however, included added layers of complexity involved keeping two simultaneous geographical efforts coordinated and tracking requirements for DCGI, The Gambia's Ethics Committee, data safety monitoring boards, WHO prequalification, and other elements. Frequent check-ins (with regular multipolar regulatory engagements) helped mitigate these challenges.
Value of partner continuity and trust. Partnering across multiple projects and similar-class products built upon expertise and enabled efficiencies. The fact that several of PNEUMOSIL's collaborators had previously worked together on vaccine development enabled learnings to be applied to PNEUMOSIL and leveraged trusting relationships. Expertise from groups like UCL, PVP's SAB, the UK National Institute of Biological Standards and Control, and consultants further strengthened the ability to develop and evaluate the vaccine successfully. Strength of partnership also helped expedite the WHO prequalification and Indian licensure process. Additionally, early large-scale and long-term financial and resource commitments from SIIPL and the Bill & Melinda Gates Foundation provided leeway needed for end-toend line of sight, therefore reducing risk and streamlining effort. The team also aligned with advocacy groups to share evidence, raise awareness, and strengthen the enabling environment for new PCVs. Overall, partner trust and continuity-conserved resources, accelerated timelines, tapped established lines of communication, reduced risk, and streamlined administrative process. (Table 4

.)
Complementary philosophies and early clarity of purpose kept project on target. Commonalities between PATH's PVP goal and SIIPL's philosophy of producing high-quality vaccines at high volume and low prices for LMIC access were critical to achieving early clarity of purpose for PNEUMOSIL. Both organizations agreed at the outset that the PCV should be affordable for children most in need. Likewise, agreeing on the value proposition, TPP, and global access principles were essential starting points that reduced downstream risk and ensured a fitfor-purpose product.

Conclusion
PNEUMOSIL's market entry is an important milestone toward enhancing the world's arsenal of state-of-the-art PCVs and alleviating the price and supply barriers that have historically hindered or precluded sustainable access for LMICs. It ends a decade-long drought in new PCV suppliers and could reshape the marketplace by providing a more affordable option of comparable quality and potential impact. For Gavi and the countries it supports, PNEUMOSIL's 30% savings over other PCVs means public health funds can go farther against pneumococcal disease and other health priorities. This more costefficient option is also important for non-Gavi-supported LMICs, who face paying for PCVs themselves, often at elevated prices (e.g., 12.83-14.50 USD per dose in non-Gavi Pan American Health Organization countries). 24 PNEUMOSIL constitutes not only a technical tour de force for SIIPL as only the third vaccine manufacturer to WHOprequalify a PCV, it marks a turning point that could lead to dramatic public health impact and help put PCVs within reach for children still without affordable access. The journey would not have been possible without a diversity of like-minded dedicated researchers, manufacturers, governments, multilaterals, regulators, laboratories, non-governmental organizations, and foundations across continents that provided input, debate, and manpower on decisions along the way8