In light of the shocking rates of disease and death brought about by the coronavirus disease 2019 (COVID-19) pandemic, vaccines have been developed and deployed in an amazingly short period of time. While most measures of vaccine efficacy depend on antibody-mediated neutralization of the virus, a new study showcases the power of an Adeno-Associated Virus-6 (AAV6) or AAV9 encoding an engineered spike protein to generate cellular immunity to produce long-term protection.
*Important notice: bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.
Background
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein is a homotrimer that mediates high-affinity binding to the host cell membrane-bound receptor angiotensin-converting enzyme 2 (ACE2). Over 90% of neutralizing antibodies are elicited by antigens in the receptor binding domain (RBD) on the S1 subunit of the spike.
New research, available as a preprint on the bioRxiv* server, has focused on the role played by T cell immunity, particularly CD8+ cytotoxic T cells, on preventing lethal infection with SARS-CoV-2 in mice infused with peptide-loaded antigen-presenting dendritic cells (DCs).
Adeno-associated viruses (AAVs) are non-pathogenic non-replicating viruses commonly used to vector genes into living cells. They have been approved in both the USA and Europe for gene delivery in human genetic diseases. However, their whole genomes are approximately 4,800 bases long, and can thus be used only for synthetic genome delivery of less than this size.
The current experiment
The Wuhan strain of SARS-CoV-2 spike protein was engineered to include a promoter gene that allows constitutive gene expression. The researchers used two such transgenes without the N-terminal domain (denoted by ΔNTD), namely, M8 and M8B. Both have eight mutational sites, of which only one, at position 614, is different in the two transgenes.
This was included to explore the role of this epitope, which is conserved in variants and has been described to be involved in antibody-dependent enhancement with SARS-CoV and higher transmissibility with SARS-CoV-2.
Other mutations include stabilizing proline insertion and mutations at the furin and S2 protease sites.
Transgenes induce T cell response
Eight mice were administered the M8B vaccine intramuscularly and assessed weekly, in groups of two, by immunohistochemistry. The transgene was expressed at high levels in skeletal muscle cells by seven days post-vaccination. At 21 days, the area was densely infiltrated with mononuclear cells, mostly macrophages and CD8+ cytotoxic T cells.
This indicates the cellular immune response to the transgene expression in the muscle fibers. Regenerating muscle cells are also found near these immune cells, indicating that these transduced muscle fibers are eliminated by the infiltrating cells. The infiltrate appeared to be specifically induced by the spike transgene since a non-immunogenic AAV9 vector did not cause macrophage or cytotoxic T cell infiltration.
T cell responses were recorded by ex vivo stimulation tests using peptide pools that contained fragments of S1 and S2 subunits of the spike. Numerous gamma-interferon (IFNγ)-secreting cells were found, typical of a robust type 1 immune reaction, following the use of AAV9 or AAV6 encoding either M8 or M8B.
Protective response against infection
Using transgenic mice that express the human ACE2 receptor, the scientists found that after vaccination with these transgene-bearing vectors, a T helper type 1 (Th1) response was found within spleen lymphocytes in response to spike peptide pool stimulation. Th2 cytokines were not significantly increased, except for IL-10, which was secreted by a few.
Thus, the cellular immune response to the spike protein was skewed towards a Th1 response, despite the mutations in the alpha variant of the spike protein.
Protection against pneumonia
The vaccinated mice were also found to be protected against symptoms of severe COVID-19, while 100% of controls met euthanasia criteria by developing such features at seven days post-immunization.
Over this period, tests showed that immunized mice maintained a stable weight, good cardiopulmonary status, and intact respiratory performance. In all cases, control animals suffered deterioration. Histology showed that the latter had gross interstitial pneumonia with alveolar space collapse.
The viral ribonucleic acid (RNA) load was reduced by 2.9-4.3 fold with the various vaccines compared to the control group, corroborating the clinical findings.
Cellular vs. humoral immunity
Despite the absence of neutralizing antibodies, the mice were highly protected against infection and lung changes. A booster after five weeks still failed to produce humoral immunity when assessed at three weeks from the second dose. By the end of the experiment, neutralizing antibodies were higher in the vaccinated challenged group vs. the unchallenged vaccinated cohort and the challenged unvaccinated group.
Thus, these primed CD4+ T cells boost neutralizing antibody production, both in magnitude and speed of response, following infection. The immunoglobulin G (IgG) titers to the RBD in challenged vaccinated mice were approximately 7-fold and 14-fold that in unchallenged vaccinated mice, with the AAV6-M8B and AAV9-M8B groups, respectively.
The researchers write:
Together, these serological studies reveal that AAV-M8/M8B vaccination alone produces a very mild humoral immune response with no detectable neutralizing antibody component; however, upon infection vaccinated mice respond with an accelerated humoral response.”
Shelf life
The vaccines were cryogenically stored and used to vaccinate mice. This vial was then stored for four months at 4oC and then used to vaccinate another set of mice. Both were then challenged with the virus approximately two weeks later.
Even at 4.5 months from a single vaccine dose, the mice were protected against infection, and the vaccine remained stable after freezing and long-term refrigeration.
A macaque study confirmed these findings with one dose of the vaccination.
What is the conclusion?
The scientists concluded that this vaccine candidate allowed vaccinated mice to survive a lethal SARS-CoV-2 challenge. Secondly, both immediate and durable T cell protection against infection with this virus, despite a remarkable absence of neutralizing antibodies, was demonstrated in mice. Thirdly, unexpected, this cellular response accelerated the affinity maturation of neutralizing antibodies to the mutant RBD irrespective of the SARS-CoV-2 variant to which the host is initially exposed.
This could bypass antigenic imprinting to the RBD epitopes of the Wuhan lineage caused by first-generation vaccines. T cell epitopes cover a range of epitopes on the spike protein sequence, unlike B cells which focus on a narrow range of conformational epitopes that are targeted by most individuals for successful neutralization.
Thus, the strength and durability of vaccine-induced T cell immunity has important implications for the future evolution of the COVID-19 pandemic as herd immunity to the original variant escalates and selective pressure builds for SARS-CoV-2 variants capable of escaping neutralizing humoral immunity through mutations in the RBD,” write the team.
*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.
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