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  1. Evaluation Summary:

    This paper describes an approach in which a non-replicating influenza virus expressing a cancer testis antigen is used to induce a systemic and mucosal antigen specific T cell responses. The authors find that this immune response is sufficient to reduce tumor burden following intravenous or subcutaneous tumor challenge. This paper is potentially interesting to tumor-immunity researchers.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

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  2. Reviewer #1 (Public Review):

    This paper is potentially interesting to many researchers who are looking for ways to enhance the CTL response to tumor immunity in combination with checkpoint antibodies.


    1. The authors engineered a non-replicating pseudotyped influenza virus to deliver the well-known cancer testis antigen, NY-ESO-1 (NY-ESO-1 S-FLU). One problem in using virus vectors for vaccination is the immune reaction to viral protein. They clearly showed that Switching HA coat of virus in S-FLU construct would easily overcome the pre-existing anti FLU immunity. This finding is new and interesting.

    2. The authors also showed that intranasal or intramuscular immunization of NY-ESO-1 S-FLU virus in mice elicited a strong NY-ESO-1 specific CD8+T cell response in lungs and spleen that resulted in the regression of NY-ESO-1 expressing lung tumor and subcutaneous tumor respectively. In addition, they demonstrated that NY-ESO-1 S-FLU synergistically works with anti-PD1, which is also interesting.


    1. Mechanisms by which NY-ESO-1 S-FLU works better than other types of viral vectors are unclear.

    2. To most readers who do not have much information on cancer vaccine, it is unclear whether the anti-tumor effect of NY-ESO-1 S-FLU was high or modest compared with those reported in the similar studies using other types of vaccines (e.g., peptide vaccine, mRNA vaccine,----).

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  3. Reviewer #2 (Public Review):

    Virus-based tumor vaccines can induce a robust T cell response capable of limiting tumor progression. The authors sought to investigate whether the influenza virus can be used to induce a protective tumor antigen-specific T cell response. They generated a replication deficient influenza virus expressing a testis cancer antigen and found that infection of mice with this virus can reduce lung or subcutaneous tumor burden depending on the route of immunization. Furthermore, the authors show that PD1 blockade can further augment the tumor protection elicited by this approach.

    One limitation of this study is that authors do not show that their approach induces long-lived anti-tumor immunity. While the authors show that their approach can induce a memory T cell response in the lungs, they do not perform any experiments in which mice are challenged at a memory time point. Rather their prophylactic tumor model challenges the mice two days after booster infection. Furthermore, their therapeutic model relies on repeat infection 14 days apart which is also not sufficient to generate a mature memory T cell response. Therefore, it is difficult to conclude from this study whether their approach will induce long-lasting immunity.

    An additional limitation of their approach is that most humans have been infected by influenza or received influenza vaccines many times in their lives. Therefore, most individuals would be expected to have some influenza-specific antibodies that can bind to conserved elements of the hemagglutinin protein. To circumvent this they showed that Switching HA coat of virus in S-FLU construct could overcome the pre-existing anti FLU immunity.

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