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Principal Investigator: paolo lusso
Organization: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
Fiscal Year: 2024
Award: $133,896
Funding agency: National Institute of Allergy and Infectious Diseases
Following the success of our HIV-1 vaccine platform based on co-formulated Env+Gag mRNA in order to produce non-infectious VLPs (Zhang et al., Nat. Med. 27:2234, 2021), in collaboration with Moderna, Inc., we have designed a similar vaccine platform for SARS-CoV-2. The major results obtained thus far include the following:
1. We synthesized different tail-modified SARS-CoV2 envelope spikes (S) in order to facilitate cognate Spike-Gag interaction and VLP formation with lentivirus core proteins. To promote the assembly of VLPs containing lentivirus (HIV-1 or SIV) core proteins, we have designed S proteins modified in their cytoplasmic tails (CT) by replacement of the natural tail with either the HIV-1 or SIV gp41 CT. A truncated form of the SIV tail (Sst) has been selected based on its ability to promote a more efficient surface membrane expression. Moderna Inc. has produced all the specific mRNAs according to our design.
2. In vitro, we tested the efficiency of production of VLPs with the modified spike proteins. The spike protein mRNAs were co-transfected into mammalian cells in all possible combinations with mRNAs encoding HIV-1 or SIV Gag or Gag-Pol. The extracellular VLPs were harvested and concentrated by ultracentrifugation on sucrose cushions. The produced VLPs have been extensively characterized both quantitatively and qualitatively using a wide array of physical and immunological assays, including virion capture by mAbs followed by ELISA for lentivirus core antigen quantification, Western blot and others.
3. In vivo, we tested the immunogenicity of our hybrid SARS-CoV-2-SIV VLP-forming vaccine in BALB/c mice. The animals were vaccinated with SSt+gag mRNA or Set alone at 0, 4 and 16 weeks. The VLP-forming vaccine (Sst+Gag) induced higher titers of Spike-binding and autologous neutralizing antibodies at all time points compared to SSt mRNA alone. Furthermore, mice immunized with SSt+gag mRNA developed neutralizing antibodies effective against different variants of concern.
These data demonstrate that the Gag/VLP mRNA platform can be successfully applied to vaccines against different agents for the prevention of infectious diseases of global relevance.
Our research on pathogenesis led to the identification of circulating anti-PF4 antibodies associated with disease severity in patients with COVID-19. The pathogenic mechanisms that trigger the most severe complications of COVID-19, which are often fatal, are still largely unknown. Severe COVID-19 is frequently associated with thrombosis and thrombocytopenia, which are reminiscent of the clinical presentation of two life-threatening syndromes: heparin-induced thrombocytopenia (HIT), which develops in patients treated with high doses of heparin after cardiac or orthopedic surgery, and vaccine-induced thrombosis with thrombocytopenia (VITT), which is a rare complication of adenovirus-vectored COVID-19 vaccines. The pathogenic mechanism of both the HIT and VITT syndromes involves the elicitation of autoantibodies that target partially cryptic epitopes in the a-chemokine platelet factor 4 (PF4 or CXCL4), which are fully revealed upon binding to heparin or other polyanionic molecules.
We postulated that the SARS-CoV-2 Spike protein might directly interact with PF4 and induce the production of anti-PF4 autoantibodies. Indeed, we obtained multiple lines of evidence that the Spike protein directly binds PF4 and forms very large multi molecular complexes, which could be immunogenic due to their very large size. Using a clinically validated immunoassay, we found anti-PF4 antibodies in 95.0% of hospitalized COVID-19 patients irrespective of prior heparin treatment. Anti-PF4 antibody levels were correlated with the maximum disease severity score and with significant reductions in circulating platelet counts during hospitalization. In individuals convalescent from COVID-19, the antibody levels returned to near-normal values. Sera from COVID-19 patients induced higher levels of platelet activation than sera from healthy blood donors, but the results were not correlated with the levels of anti-PF4 antibodies. These results demonstrate that the vast majority of patients with severe COVID-19 develop anti-PF4 antibodies, which may play a role in the clinical complications of COVID-19, and have implications for the therapeutic management of the most severe cases.
To further validate the role of anti-PF4 autoantibodies in COVID-19 pathogenesis, we attempted to generate a small animal model in mice. Thus, we modified a previously reported mouse model for the induction of HIT autoantibodies to mimic the pathogenic mechanism of COVID-19. Mice were immunized daily for 5 days with molecular complexes of the SARS-CoV-2 Spike protein and murine PF4. As a control, we used the traditional complexes of PF4 with HMW heparin. Although the titers remained relatively low, we did observe the elicitation of serum HIT-like antibodies, as detected by ELISA against PF4-heparin complexes. Further studies are underway to better characterize these antibodies and clone monoclonal antibodies with HIT-like characteristics.
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