S-pseudotyped VLPs offer promising COVID-19 vaccine platform

With the continuing COVID-19 pandemic, several researchers have brought out candidate vaccines, some of which have undergone human testing. However, it is unclear whether they are genuinely protective and how long this immunity lasts. A new study published on the preprint server bioRxiv* in September 2020 reports on a Moloney murine leukemia virus (MLV) platform that produces VLPs expressing the important spike antigen of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

Viral Vaccine Platforms

The primary vaccine target for SARS-CoV-2 is the spike or S protein, which mediates viral engagement with the host cell receptor, the human angiotensin-converting enzyme 2 (ACE2). Many platforms are currently available for vaccine delivery, namely, RNA, DNA, recombinant proteins, live attenuated virus (LAV) and inactivated viruses, viral vectors, or virus-like particles (VLPs).

Advantages of VLPs

While RNA, DNA, and protein vaccines are easier to manufacture, those derived from VLPs or LAVs are more effective at inducing an immune response sustained over a more extended period. This is an attractive aspect concerning a potential COVID-19 vaccine expected to elicit a durable neutralizing antibody (NAb) reaction.

Earlier studies indicate that vaccination in humans leads to NAb titers at least comparable to that assayed in convalescent serum following natural COVID-19 infection. Recently, a DNA vaccine study indicated the persistence of immunity to the virus for at least 13 weeks in immunized monkeys.

VLPs are generated by assembling viral proteins that lack genetic material and are therefore not able to replicate, but elicit a high degree of immunity since they are rapidly recognized by antigen-presenting cells. The surfaces of these particles bear a repeating array of viral antigens that leads to the induction of both innate and adaptive immunity, with high neutralizing activity.

These have been used to deliver vaccine antigens in the case of human papillomavirus (HPV), hepatitis, and influenza viruses. These vaccines can also be lyophilized, making storage and distribution of the vaccine much more straightforward, enabling broader coverage in the current situation where the entire world requires vaccine protection.

VLP Platform for COVID-19 Vaccine

The current study reports the production and characteristics of a VLP based on the Moloney murine leukemia virus 92 (MLV). The coronavirus is assembled within the protein-synthesizing machinery of the cytoplasm, which made it necessary to assay the presence of the S protein at the surface. They found that both the full-length S protein as well as a shorter version of the S protein was trafficked to the surface of the cell, which is essential to its immunogenicity.

Infectious MLV Recombinants Produced Inefficiently and Transiently

After expressing the SARS CoV-2 S protein, the researchers looked at the titer of pseudotyped VLP expressing the S protein. They found that the production of infectious VLPs was very low and transient.

DS-Pseudotyped Vectors Produced Efficiently

Secondly, they found that the pseudotyped MLV particles expressing the deletion-carrying S (DS) antigen, but not those expressing the S antigen, are released effectively from the producer cells at 10 times or more the titer of the transiently transfected particles, provided a better cell system was used.

Deletion of S Tail Not Fusogenic

The researchers also observed that when the 19-amino acid cytoplasmic tail of the spike protein was deleted, the fusogenicity remained unchanged. This shows that there is another reason for the high transduction efficiency of the DS-pseudoviruses.

The MLV VLPs Incorporate High Amounts of DS

In order to arrive at a stable production of a vaccine, the VLPs must produce high amounts of S protein at their surface. The researchers did a quantitative evaluation of S protein in the VLPs generated transiently as well as from stable producer cells. They found that the level of S and DS production in stable producer cells is 4 and 15 times higher than in transient transfection.

They found evidence that dimeric and trimeric forms of DS protein were produced at much higher levels than with the full-length S protein. The S and DS levels in cellular extracts varied by only 1.5-fold. Thus, MLV VLPs incorporate DS very efficiently in stable producers.

MLV VLPs Preferentially Incorporate SARS-CoV2 DS protein

The researchers also examined whether the DS protein was incorporated into the VLPs or into extracellular vesicles. They found that it was preferentially found in the former.

The researchers suggest that, based on these findings, the MLV VLP platform they established is suitable for the production of a vaccine against COVID-19. The efficient incorporation of the S antigen is necessary to produce a reliable platform.

Reason for Higher Efficiency of DS

Prior studies suggested that the optimization of the spike protein codon and the deletion of the ER retention signal in the cytoplasmic tail are useful for enhancing the efficiency of pseudotyping of MLV, HIV, SIV, and VSV vectors.

While the optimization of the S codon upregulates S expression, the contribution of the deletion of the tail segment was unknown. Some researchers reported no change in S expression when this underwent a missense mutation. This is confirmed in the current study, where S detection at the cell surface was comparable to DS detection in both transient transfection and stable producer cell lines.

The researchers conclude that S can migrate efficiently to the cell surface under conditions of overexpression. Even then, DS achieved higher levels of incorporation into the MLV VLPs.

The reason for the difference is not in the enhancement of pseudotyping produced by the reduction of the steric hindrance of the retroviral matrix proteins, as previously proposed. This is shown by the detection of increased DS in transfected cells that lack these proteins.

Instead, it may be that due to the close similarity in the composition of VLPs and EVs, they use the same type of trafficking pathways. This explains why DS but not S is efficiently taken up by both of these particles, similar to endosomal markers.

This also means that the small proportion of EVs (<10%) released from transiently transfected cells are just as highly immunogenic as the VLPs and can be left in the preparation. In fact, EVs incorporating SARS-CoV S are found to elicit high levels of NAbs.

Viral Titer Increases Thousand-Fold with DS

The thousand-fold increase in the viral titer generated from stable producers with DS compared to S incorporation belies the only 15-fold difference in the amount of the two proteins at the surface of the VLP. This is not due to higher fusogenicity, as shown in this study.

Instead, the researchers suggest that “recombinant viruses become fully infectious when a certain threshold of S protein is incorporated at their surface.”

Implications and Future Directions

The study made use of recombinant GFP or luciferase pseudotyped retroviruses to measure NAb activity in immune serum, because these are convenient to use, requiring only BSL-2 conditions. This could be used to detect NAbs in larger cohorts.

An earlier study suggested that microgram doses of a nanoparticle vaccine containing the S antigen elicited high NAb titers. Based on this, the researchers suggest that this VLP-based platform could be as efficient with comparable or lower amounts of the S protein. The use of optimal producer cell lines and bioreactor conditions could enhance the yield, as has been shown in gene therapy vector experiments, where the yield of the vector and the average titer went up by 13-fold and ~6-fold in a bioreactor as compared to a ten-layer cell factory.

Finally, the use of VLPs could be useful for immunogenicity because they express a recombinant antigen format, which has been shown to be more immunogenic than the wildtype protein alone. This scalable platform could be used to produce broad-spectrum coronavirus vaccines on a large scale. Such nanoparticles could be used in COVID-19 either as a stand-alone vaccine or boost the immune response to other vaccines.

*Important Notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.