UC Santa Cruz

A major focus of HIV vaccine development has been improving the envelope glycoprotein 120 (gp120) derived immunogens used in the RV144 HIV vaccine trial. RV144 has been the only human clinical trial to demonstrate modest but significant protection in humans. Over the last 30 years it has been difficult to manufacture Clade B immunogens, such as MN-rgp120 used in the RV144 trial. Clade B immunogens are representative of viruses from North America, Europe and Australia and become proteolyzed when expressed in Chinese Hamster Ovary cells. In this report, we identified complement component 1s (C1s) as the protease responsible for cleavage in the third variable region (V3) at a site recognized by neutralizing antibodies. To solve this problem, we used CRISPR/Cas9 to inactivate the C1s-gene of several CHO cell lines including the standard CHOS, CHOK1 cell lines and the glycan restricted MGAT1- CHO cell line. These novel cell lines provide the means to produce Clade B immunogens as well as other recombinant proteins without proteolysis. Proteolysis of recombinant proteins is a common problem in the biopharmaceutical industry. Another medically significant protein that also becomes proteolyzed during expression in CHO cells is human Factor VIII (FVIII). Biochemical analysis was carried out to see if unintended cleavage of FVIII could also be attributed to C1s. Differences between the substrate specificity of human thrombin and CHO C1s were elucidated using mutagenesis and homology modeling and showed that FVIII proteolysis was due to another protease. In other studies, we were interested in enhancing the immunogenicity of epitopes in the second variable region (V2) of A244 gp120 that correlated with protection in the RV144 clinical trial. This was accomplished through the characterization of 10C10, a mouse monoclonal antibody elicited by immunization with A244-gp120. 10C10 was shown to have cross-reactivity to clade AE, B and C gp120s and bind to a V2 peptide through protein- and peptide-binding studies. In the RV144 trial, non-neutralizing antibodies to the V2 region were found to be a correlate of protection. Similar to the CH58-like antibodies that have been identified, 10C10 was found to be non-neutralizing and bind to a V2 peptide that assumed an alpha-helical conformation as determined by computational modeling. Preventing proteolysis and targeting antibodies to the 10C10 site will help guide the development of the next generation of vaccine candidates to achieve the goal of making a viable HIV vaccine.