T cell-mediated protection against lethal SARS-CoV-2 challenge by a synthetic long peptide vaccine targeting conserved CD4+ and CD8+ T cell epitopes

Technical Summary: T Cell-Mediated Protection Against Lethal SARS-CoV-2 Challenge by a Synthetic Long Peptide Vaccine

Problem Statement
The majority of approved SARS-CoV-2 prophylactic vaccines rely on mRNA or viral vector platforms to induce neutralizing IgG antibodies against conformational B cell epitopes of the viral Spike (S) protein. While effective, these antibody-dependent strategies have limitations: they are less protective in patients with hereditary or acquired B cell deficiencies (e.g., those receiving anti-CD20 therapy) and may be compromised by antigenic drift in the Spike protein. Furthermore, severe COVID-19 is often characterized by impaired T cell immune responses. There is a critical need for vaccination strategies that elicit robust, virus-specific T cell immunity to protect individuals with defective B cell immunity and to provide resilience against viral variants.

Methodology
The study developed "Corona-SLP," a synthetic long peptide (SLP) vaccine designed to target conserved regions of the SARS-CoV-2 Spike (S), Nucleocapsid (N), and Membrane (M) proteins. The development process involved:

1. In Silico Design and Selection:

   • Immunogenicity Prediction: A proprietary tool, ISABELLA (a Logistic Regression model trained on MHC binding, proteasomal cleavage, and TAP transport data), was used to identify immunogenic regions for frequent HLA types.
   • Manufacturability Filtering: A proprietary Random Forest model, MONALISA, predicted synthesis success rates to filter candidates.
   • Initial Pool: 45 SLPs were selected, reduced to 21 based on synthesis feasibility, and finally refined to 11 based on ex vivo validation.
   • Coverage: The final set covers 99.915% of HLA-I and 100% of HLA-II epitopes in European and North American populations.

2. Ex Vivo Human Validation:

   • Peripheral Blood Mononuclear Cells (PBMCs) from 11 SARS-CoV-2 convalescent donors and 4 healthy controls were screened via IFN-γ ELISpot and intracellular cytokine staining.
   • Candidates were evaluated for their ability to reactivate memory CD4+ and CD8+ T cell responses.

3. Murine Model Optimization (mCorona-SLP):

   • A murine analogue (mCorona-SLP) was created, combining human epitope-based SLPs (immunogenic in C57BL/6 mice) with validated C57BL/6-specific SLPs.
   • Parameters Tested: Administration routes (intradermal, subcutaneous, intramuscular), adjuvants (Amplivant, a TLR1/2 ligand, vs. Montanide), dosing (10 µg vs. 50 µg per SLP), and vaccination intervals (2 vs. 3 weeks).
   • Adjuvant: Amplivant was used to conjugate SLPs to enhance dendritic cell maturation and T cell priming.

4. In Vivo Protection Studies:

   • Models: K18-hACE2 transgenic mice (highly susceptible) and human ACE2 knock-in mice (moderately susceptible).
   • Challenge: Mice were challenged with a lethal dose of SARS-CoV-2 (Leiden-08 isolate) intranasally.
   • Endpoints: Survival, weight loss, clinical scores, lung viral load (RNA and plaque assay), and pseudovirus neutralization titers.

5. Variant Analysis:

   • In silico analysis assessed the impact of five Variants of Concern (Alpha, Beta, Delta, Gamma, Omicron) on the selected epitopes.

Key Results

• Human Memory Reactivation: The selected Corona-SLPs successfully reactivated robust memory CD8+ T cell responses in PBMCs from convalescent donors. SLP-14 elicited the highest response frequency. The vaccine induced a Th1-biased response (high IL-1β, low IL-4/IL-5). While some SLP-specific IgG was detected, it was often low or cross-reactive, and no neutralizing antibodies were induced by the SLPs alone.
• Optimization of Murine Strategy:
  • Route: Intradermal administration combined with Amplivant yielded superior antigen-specific CD8+ and CD4+ T cell responses compared to subcutaneous or intramuscular routes.
  • Adjuvant: Amplivant was essential for efficient T cell priming; vaccination without adjuvant failed to induce detectable IFN-γ responses.
  • Dosing and Interval: 50 µg per SLP with 50 µg Amplivant at 2-week intervals produced the highest T cell frequencies.
  • Antigenic Competition: While single SLP vaccination induced slightly higher circulating T cell frequencies for specific epitopes, the multi-SLP formulation induced a broader response (covering multiple epitopes) and robust CD4+ help, which was absent in single-SLP vaccinated mice.
• In Vivo Protection:
  • Survival and Morbidity: Prophylactic vaccination with mCorona-SLP (11 SLPs + Amplivant) protected K18-hACE2 mice from lethal challenge, resulting in minimal weight loss, lower clinical scores, and 100% survival. In contrast, mice vaccinated with a single SLP (mSLP2) or unvaccinated controls succumbed to infection.
  • Viral Load: In hACE2 knock-in mice, mCorona-SLP vaccination significantly reduced lung viral RNA and infectious virus titers compared to controls.
  • Mechanism: Protection occurred in the absence of neutralizing antibodies (pVNT50 was undetectable), indicating that the protection was mediated primarily by vaccine-induced T cell responses.
• Variant Resilience: In silico analysis showed that the conserved regions targeted by the vaccine are largely preserved across all five major Variants of Concern. Only one or two SLPs per lineage were affected by mutations, suggesting the vaccine's immunogenicity would be retained across variants.

Significance and Claims
The paper claims that the Corona-SLP vaccine demonstrates that targeting conserved SARS-CoV-2 epitopes with synthetic long peptides can induce robust, protective T cell immunity. The study highlights that this protection is effective even in the absence of neutralizing antibodies, supporting the utility of T cell-focused vaccination for populations with impaired B cell immunity (e.g., elderly or immunocompromised patients). The findings suggest that SLP vaccines offer a potential solution for variant-resilient immunity by targeting conserved regions of the S, N, and M proteins, rather than the highly mutable Spike protein alone. The authors conclude that this approach warrants further evaluation, particularly for settings where antibody-mediated immunity is limited or compromised.