A Review of Protein-Based COVID-19 Vaccines: From Monovalent to Multivalent Formulations

The emergence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), resulting in the COVID-19 pandemic, has profoundly impacted global healthcare systems and the trajectory of economic advancement. As nations grapple with the far-reaching consequences of this unprecedented health crisis, the administration of COVID-19 vaccines has proven to be a pivotal strategy in managing this crisis. Protein-based vaccines have garnered significant attention owing to their commendable safety profile and precise immune targeting advantages. Nonetheless, the unpredictable mutations and widespread transmission of SARS-CoV-2 have posed challenges for vaccine developers and governments worldwide. Monovalent and multivalent vaccines represent two strategies in COVID-19 vaccine development, with ongoing controversy surrounding their efficacy. This review concentrates on the development of protein-based COVID-19 vaccines, specifically addressing the transition from monovalent to multivalent formulations, and synthesizes data on vaccine manufacturers, antigen composition, pivotal clinical study findings, and other features that shape their distinct profiles and overall effectiveness. Our hypothesis is that multivalent vaccine strategies for COVID-19 could offer enhanced capability with broad-spectrum protection.


Introduction
Since the World Health Organization (WHO) reported the first case of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection on 31 December 2019 [1], the WHO has documented more than 774 million confirmed COVID-19 cases and more than 7 million deaths as of 18 February 2024 [2].In contrast to measures, like public transport closures, remote work, and full lockdowns, implemented for epidemic control, vaccination has emerged as a proven cost-effective and efficient strategy against the COVID-19 pandemic [3][4][5].
A variety of platforms, including inactivated virus, viral vectors, protein-based vaccines, and innovative mRNA vaccines, are currently used in COVID-19 vaccines, either licensed or in development [6].While the administration of COVID-19 vaccines has significantly reduced mortality, severe disease, and overall disease burden, thereby facilitating the reopening of societies, evidence over the past three years suggests that the virus is continuously evolving, leading to the emergence of new variants or sub-lineages in the future [7].Concerns about waning immunity over time, increased transmissibility, and immune escape due to the evolving SARS-CoV-2 variants persist [8,9].The emergence of the SARS-CoV-2 Omicron JN.1 variant was initially identified in the United States in September 2023 and was the predominant variant in the country until 23 December 2023.Concurrently, the incidence of COVID-19-related hospitalizations has shown an upward trend since 4 November 2023, presenting a significant challenge to currently approved COVID-19 vaccines.
Thus, in this review, we analyzed the dynamic antigen compositions of proteinbased COVID-19 vaccines authorized by various regulatory authorities, including the Food and Drug Administration (FDA), the European Medicines Agency (EMA), and the National Medical Product Administration (NMPA) in China.Additionally, we provided a comprehensive summary of the efficacy and immunogenicity of both monovalent and multivalent protein-based COVID-19 vaccines against both antigen-matched and antigenmismatched variants.

Protein-Based COVID-19 Vaccines
Recognized as one of the safest and most extensively utilized vaccine platforms, protein-based vaccines have demonstrated high efficacy in preventing diseases, such as hepatitis B and C, influenza, pertussis, and human papillomavirus [23].The antigens of protein-based vaccines are usually recombinant proteins, which were generated by various cell-expressing systems, including bacteria, yeasts, insects, and mammalian cells, using viral proteins or peptides as their basis [23].Administrated with an appropriate adjuvant, these recombinant proteins tend to provide a more robust and durable immunization.
Though they present prominent advantages, protein-based vaccines pose several challenges, including the possible need for adjuvants and multiple inoculations, which can increase the risk of adverse reactions [24].The complex manufacturing process, with its high costs and specialized requirements, may restrict scalability and accessibility, particularly in resource-limited settings [25].Furthermore, variations in antigen choices, adjuvant incorporation, and individual immune system responses can yield inconsistent vaccine effectiveness across different populations.Specifically concerning SARS-CoV-2 vaccines, those primarily focusing on the receptor-binding domain (RBD) of the spike protein might lack the epitope diversity present in the full-length spike, potentially reducing their efficacy against emerging viral variants [26].In response to the relentless evolutionary dynamics of the novel variants or subvariants of SARS-CoV-2, characterized by the emergence of new variants or subvariants with distinct mutations, the antigenic composition of the vaccine has been strategically adapted and refined to align with these shifting molecular landscapes (Figure 1; publications related to the key clinical studies are summarized in Table 2).These vaccines have demonstrated the ability to elicit a robust neutralizing antibody (nAb) response and significant Th1 and Th2 cell responses against SARS-CoV-2 and its variants.Furthermore, they exhibit safety and reactogenicity profiles that are favorable and comparable to conventional inactivated COVID-19 vaccines [51].Taking the advantage of thermostability, protein-based vaccines can be stored and transported at temperatures ranging from 2 to 8 • C [52].This characteristic ensures their accessibility in resource-limited regions and provides a cost-effective solution for widespread distribution.[53][54][55].It contains the full-length S protein of the prototype strain and the adjuvant Matrix-M, triggering a strong immune response, involving both B and T lymphocytes against the SARS-CoV-2 S protein [56,57].
Authorization for Nuvaxovid was based on data derived from two phase 3 and one phase 2 clinical studies (Table 3) [29,30].The first phase 3 study, conducted in the United Kingdom, involved 14,039 adults aged over 18 years who were negative for SARS-CoV-2 infection at baseline.Participants received two doses of Nuvaxovid or placebo, given 21 days apart [29].The vaccine showed 89.7% efficacy against symptomatic SARS-CoV-2 infection seven days after the second dose.Post hoc analysis also found vaccine efficacy of 86.3% against the Alpha SARS-CoV-2 variant and an impressive 96.4% vaccine efficacy against non-Alpha SARS-CoV-2 variants, primarily the prototype strain (Table 4) [29,58].The definition of symptomatic SARS-CoV-2 infection may be different between the various COVID-19 vaccines; a direct comparison of vaccine efficacy would be misleading.† Mean time for efficacy follow-up.
The second phase 3 study, conducted in Mexico and the United States, enrolled 29,582 participants aged over 18 years with no previous SARS-CoV-2 infection.Participants were randomized at a 2:1 ratio to receive two doses of Nuvaxovid or placebo, with a 21-day interval between doses [30].The vaccine demonstrated an efficacy of 90.4% against reverse transcriptase-polymerase chain reaction (RT-PCR)-confirmed SARS-CoV-2 infection seven days after the second vaccine dose [30].Notably, Nuvaxovid exhibited an efficacy of 93.6% against the Alpha variant and 92.6% against all variants of concern (VOCs) or variants of interest (VOIs), including Alpha, Beta, Gamma, Epsilon, Iota, Kappa, and Zeta (Table 4) [30].Following the completion of two phase 3 studies outlined above, Nuvaxovid received EUA for individuals aged 18 years or older.Subsequently, in the primary extension series, 2247 adolescents aged 12 to 17 years were assigned to receive either two doses of Nuvaxovid or saline placebo, administered with a 21-day interval between doses [32].With a median efficacy follow-up period of 2 months, Nuvaxovid exhibited an efficacy of 79.5% (20 COVID-19 cases were reported, 6 in the Nuvaxovid group, and 14 in the placebo group).Notably, all sequenced viral genomes were identified as the Delta variant, showcasing an efficacy of 82.0% (Table 4) [32].Consequently, based on the findings of the extension study, Nuvaxovid was granted approval for individuals aged 12 to 17 years.
Considering the waning immunogenicity and efficacy of COVID-19 vaccines over time, the administration of booster doses became imperative.The immunogenicity of Nuvaxovid as a first booster dose was assessed in healthy adults aged 18 years or older.In an ad hoc analysis of PREVENT-19 (NCT04611802), 298 participants received a single booster dose of Nuvaxovid at least 6 months following completion of the initial two-dose regimen [33].This study revealed that humoral responses remained robust, regardless of the interval between primary series and booster vaccinations, although an extended interval led to enhanced responses.Additionally, increased immune responses against the Omicron BA.1, BA.2, and BA.5 variants were noted following booster shots compared to post-primary vaccinations in a subset of 14-18 participants tested [33].
A UK Phase 2 trial enrolled 2878 people aged 30 years and above who had received two doses of ChAdOx1 nCov-19 or BNT162b2.They were randomized to receive full or half doses of Nuvaxovid, other vaccines, or a placebo [34].Nuvaxovid, given as a heterologous booster, significantly raised antibodies against the initial SARS-CoV-2 virus.Consequently, Nuvaxovid was approved as a first booster, to be given at least six months post-primary COVID-19 vaccination for those aged 18 years and above.Common side effects-mostly mild to moderate and short-lasting, resolving within days-included injection site reactions, fatigue, muscle and joint pain, headache, and gastrointestinal issues [54].
The emergence of SARS-CoV-2 XBB subvariants prompted a need for vaccine updates due to waning immunity.Heeding advice from VRBPAC and TAG-CO-VAC, the 2023-2024 COVID-19 vaccine formulation was recommended to focus on XBB lineages [11,12].Novavax swiftly developed a monovalent protein-based vaccine derived from NVX-CoV2373, specifically targeting the XBB.1.5subvariant, presenting the full-length spike protein in its native form with the Matrix-MTM adjuvant [35].This formulation robustly stimulates neutralizing antibodies against multiple XBB subvariants and fosters Th1-skewed CD4 + T-cell responses in animal models, including those previously vaccinated with different formulations [35].Based on these findings, the US FDA issued an Emergency Use Authorization for the Novavax XBB.1.5Vaccine for individuals 12 and older on 3 October 2023 [60].

V-01
Livzon Mabpharm's V-01 vaccine utilizes a unique design, incorporating the original SARS-CoV-2 RBD in a dimer-IFN-Pan Fc fusion.This design stimulates dendritic cell migration to lymph nodes, enhancing antigen presentation.Preclinical mouse trials showed that low-dose IFN-PADRE-RBD-Fc (I-R-F) triggered a strong CD8 + T-cell response and robust antibody response, demonstrating its immunogenic potential against the RBD monomer [66].In a study of 10,863 vaccinated adults in Pakistan and Malaysia (Table 3), participants were randomized 1:1 to receive V-01 or a placebo.By 27 January 2022, V-01 achieved a 47.8% efficacy against symptomatic COVID-19 from 14 days post-vaccination, fulfilling success criteria (Table 4) [39], with 79.9% efficacy against Delta and 47.0% against Omicron (Table 4) [39].Based on these results, V-01 gained EUA in China on 3 September 2023.The predominant solicited local side effect was injection-site pain, mostly mild to moderate in severity.

Vidprevtyn Beta
On 10 November 2022, the EMA fully approved Vidprevtyn Beta (CoV2 preS dTM-AS03 (B.1.351)),a monovalent booster vaccine for those aged 18 and older [67].This recombinant protein subunit vaccine, manufactured using a baculovirus system, features the B.1.351SARS-CoV-2 spike protein sans transmembrane domain and with a T4 foldon trimerization sequence.It incorporates the AS03 adjuvant, made up of squalene, DL-αtocopherol, and polysorbate 80 [64].Efficacy assessments for Vidprevtyn Beta were derived from two immunobridging trials comparing its immune response with that induced by Comirnaty (Pfizer-BioNTech mRNA vaccine), a licensed COVID-19 vaccine (Table 3).In the VAT00013 study, Vidprevtyn Beta was used as the booster injection following initial vaccination with a COVID-19 mRNA vaccine.Pseudovirus neutralization assays indicated higher geometric mean titers (GMTs) of nAb against Omicron BA.1, BA.4/5, and D614G at both Day 28 and 3 months post-vaccination, compared to Comirnaty.The Geometric Mean Titers Ratio (GMR) of Vidprevtyn Beta relative to Comirnaty on Day 28 was 2.53 and 2.50 against BA.1 and B.4/5 strains, respectively (Table 5).At month 3, the GMR of Vidprevtyn Beta relative to Comirnaty was 2.06 and 2.48 against BA.1 and BA.4/5 strains, respectively [40].In the VAT00002 study, Vidprevtyn Beta was given as a booster injection in participants primed with various types of COVID-19 vaccines (Table 3).Fourteen days after vaccination, the GMR of the Vidprevtyn Beta booster relative to the pre-booster against the B.1.351strain ranged from 38.5 to 72.3, and ranged from 14.4 to 28.6 for the D614G strain [64].The most common adverse reactions observed with Vidprevtyn Beta in the studies were pain at the injection site, headache, myalgia, malaise, arthralgia, and chills.Most adverse reactions were mild to moderate in severity and occurred within 3 days following vaccination [64,67].

Bimervax
The antigenic component of Bimervax comprises a SARS-CoV-2 virus recombinant spike (S) protein RBD fusion heterodimer, encompassing the Alpha and Beta strains, synthesized by recombinant DNA technology, utilizing a plasmid expression vector in a CHO cell line.Adjuvanted with SQBA, the immunogenicity of Bimervax was assessed in two multicenter clinical trials.In a pivotal phase 2b study, 765 participants, previously fully vaccinated with the mRNA vaccine, received a single dose of Bimervax (n = 513) or Comirnaty (n = 252) (Table 3) [65].Post-vaccination, Bimervax elicited robust production of nAb against the D614G, Beta, Delta, and Omicron BA.1 variants, with respective GMTs of 1953.89,4278.92,1466.65, and 2042.36 recorded 14 days post-injection, respectively.The GMRs of Comirnaty/Bimervax were 0.62 against Beta and 0.60 against Omicron BA.1, meeting the pre-specified criteria for Bimervax's superiority over Comirnaty (Table 5).Regarding the Delta variant, the GMT was 1466.65, and the associated GMR of Comirnaty/Bimervax was 1.02, meeting the pre-specified criteria for Bimervax non-inferiority to Comirnaty (Table 5) [65].In a single-arm study, the immunogenicity of Bimervax was evaluated among 2646 participants previously fully vaccinated with various COVID-19 vaccines, including Comirnaty, Spikevax, and Ad26.COV2-S (Table 3).Stratified by their previously received COVID-19 vaccines, the GMTs of nAb against the D614G, Beta, Delta, and Omicron BA.1 variants were comparable between the Comirnaty and Spikevax subgroups, while numerically lower GMTs were observed in the Ad26.COV2-S subgroup (Table 5) [50,65].Based on these compelling data, the EMA issued EUA for BIMERVAX as a booster for active immunization against COVID-19 in individuals aged 16 years and older who had previously received a mRNA COVID-19 vaccine on 30 March 2023 [73].The most frequently reported adverse reactions included injection-site pain (82.2%), headache (30.2%), fatigue (30.9%), and myalgia (20.2%).The median duration of local and systemic adverse reactions was 1 to 3 days, with the majority being mild to moderate and occurring within 3 days post-vaccination [65].

Future Strategies Dealing with Constantly Emerging SARS-CoV-2 Variants
To address the challenges posed by SARS-CoV-2 evolution, the Centers for Disease Control and Prevention (CDC) collaborates with others to monitor wastewater for the virus, enabling the tracking of changes in new SARS-CoV-2 variants and subvariants.This proactive approach enables communities to take swift action to prevent the spread of the infections [76].Additionally, the WHO has established the TAG-CO-VAC to review and assess the public health implications of emerging SARS-CoV-2 VOCs on COVID-19 performance.The group also provides recommendations to the WHO on COVID-19 vaccine composition [77].
Recognizing the immune-evasive nature of XBB descendant lineages, both TAG-CO-VAC and VRBPAC recommended a mono-antigen composition for the 2023-2024 COVID-19 vaccine formulation, focusing on the Omicron XBB.1.5subvariant spike protein [11,12].Nevertheless, given uncertainties about the timing, specific mutations, and antigenic characteristics of future variants, TAG-CO-VAC also noted that alternative formulations and platforms capable of eliciting robust neutralizing antibody responses against XBB descendant lineages should be considered.Developing multivalent COVID-19 vaccines with careful antigen selection and combinations may also offer a viable solution.This strategy is supported by favorable evidence from multivalent vaccines, such as SCTV01E, SCTV01E-2, Recombinant COVID-19 Trivalent Protein Vaccine (XBB + BA.5 + Delta) (Sf9 Cell), and Novel Recombinant COVID-19 Bivalent Vaccine (Original/Omicron XBB), which showed broad cross-neutralization against current and future variants and subvariants.

Conclusions
As SARS-CoV-2 continues to evolve, ongoing surveillance and adaptation of vaccine formulations will be essential to ensure continued effectiveness against emerging variants.Protein-based COVID-19 vaccines stand out as a compelling option due to their robust immunogenicity and efficacy, coupled with cost-effective storage and transportation conditions, making them potentially one of the most effective vaccine platforms.Considering the uncertainty and coexistence of various variants and subvariants of SARS-CoV-2, it is essential to develop a COVID-19 vaccine with broad and robust immunogenicity.Each variant contributes unique neutralizing epitopes, thereby broadening the spectrum of neutralizing antibodies.Moreover, certain mutation peptides observed across multiple variants are likely to persist in future emerging strains, potentially enhancing cross-reactivity with new variants.For instance, the Alpha variant exhibits the highest identity rate with the Omicron variant (99.63%) [78].Mutations, such as T95I, G142D, K417N, T478K, N501Y, P681H, delta69/70, and delta145, are shared among the Alpha, Beta, Delta, Gamma, or Omicron variants and are linked to increased transmissibility [79].Therefore, multivalent vaccines with careful antigen selection present a promising strategy to address the rapid evolution of SARS-CoV-2.

Table 1 .
Vaccine information and approved indications in FDA/EMA/NMPA.

Table 2 .
Summary of key published papers for the approved protein-based COVID-19 vaccines.