Development and Immunologic Characterization of Multi-Antigen Expressing Attenuated Poxviruses for Immunotherapy of CMV Infection in HSCT Recipients.
Authors: Diamond Don J. , Wang Zhongde , La Rosa Corinna , Grigoleit Goetz U. , Mandarino Angelo , Lacey Simon F. , Ludwig Holger , Britt William J. , Sutter Gerd , Einsele Hermann 
Published: April 2020

Abstract
CMV infection is an important risk factor in the post-transplant (Tx) recovery phase for HSCT recipients. Despite significant advances in formulation and delivery of anti-virals, their use complicates and extends post-Tx recovery and risk for CMV disease. Considering these caveats, we are pursuing a novel therapeutic strategy that focuses on priming or enhancing CMV-specific adaptive immunity in HSCT donors and recipients. 3 generations of attenuated poxvirus vectors (modified vaccinia Ankara or MVA) have been constructed to evaluate the minimal required antigenic targets to attain successful control of CMV infection. The 1st generation vector strongly expresses both pp65 and IE1, and it has been successfully demonstrated to elicit potent recall CD4 T-help and CD8 CTL responses in PBMC from CMV-positive healthy volunteers as well as stimulate primary cellular immune responses in humanized HLA transgenic mice, simultaneously for both antigens. Towards the goal of using this vector for adoptive immunotherapy, MVA infection was investigated and found to be permissive for immature myeloid DC generated with GM-CSF and IL4, as well as DC matured with TNF-α. High level expression of pp65 and IE1 insert antigens was confirmed by Western Blot (WB) for both DC populations. MVA-infected DC were also found to be efficient APC to stimulate recall CTL and CD4 T-help from CMV-positive healthy donors. The recombinant MVA, after multiple rounds of passage is stable by the criteria of antigen expression and immunogenicity. This vector is a strong candidate for clinical evaluation based on the observed protective function of pp65 and IE1 CTL in clinical trials. It is currently being manufactured under cGMP for clinical development in Germany. A 2nd generation vector that co-expresses glycoprotein gB has also been constructed, and WB confirms high level expression of each antigen. Immunologic evaluation of this vector to stimulate both pp65 and IE1-specific CTL and CD4 T-help in PBMC from CMV-positive research subjects shows similar robust stimulation as the first generation vector. In addition, studies in humanized mice demonstrate simultaneous attainment of both cellular immunity to pp65 and IE1 while humoral immunity to CMV-gB that neutalizes CMV strains in vitro was also elicited. A 3rd generation virus has been constructed which co-expresses five CMV antigens: the tegument proteins pp65 and pp150, the nuclear antigens IE1 and IE2 as a fusion gene and the polymerase subunit UL44. Immunogenicity studies of these gene products will be presented that shows simultaneous stimulation of both CTL and CD4 T-help populations from the PBMC of healthy research subjects. This vector has also been shown to prime de novo cellular immunity to all of the insert antigens in HLA transgenic mice. This vector expresses immunodominant antigens that cover &gt;90% of HLA types in most ethnic populations and would serve as the broadest and most inclusive candidate vaccine for all ethnicities. Clinical testing of these vectors as vaccines or for adoptive immunotherapy may reveal whether a requirement for greater than 2 antigens is necessary for successful limitation of viremia and disease in HSCT recipients.