Measurement of neutralizing antibodies is important for the understanding of the mechanisms used by HIV-1 to escape the host's immune system and provides critical information on the relative effectiveness of candidate vaccines. A number of neutralizing antibody assay systems have been developed, including the more traditional use of uncloned viral stocks to infect human PBMCs and more recent development of pseudotyped viruses to infected standard target cell lines (Mascola, 2004
; Mascola et al., 2005
; Montefiori, 2004
; Montefiori et al., 1998a
; Montefiori et al., 1996
; Montefiori et al., 1998b
; Ochsenbauer and Kappes, 2009
; Polonis et al., 2008
; Polonis et al., 2009
; Schweighardt et al., 2007
). The pseudotyped virus system greatly improved the reproducibility of such assays and more importantly, provided better understanding on the antigen-antibody relationship between HIV-1 Env and neutralizing antibodies.
In the current report, a modified HIV-1 Env expression vector was developed for the production of chimeric Env proteins that incorporated various primary gp120 antigens on a common gp41 background from the SF162 isolate. These results demonstrated that this modified vector, SF162-Z, was effective in expressing two heterologous primary gp120 antigens, one from clade B and one from clade C. Pseudotyped viruses incorporating these chimeric Env antigens were able to infect targeted TZM-Bl cells and these viruses are useful in the study of neutralizing antibody activities of animal immunized sera and sera from patients infected with HIV.
Previous studies have also produced chimeric Env expression vectors, such as the chimeric antigens expressing key HIV-1 Env epitopes on the backbone of HIV-2 Env (Wei et al., 2003
) and the SF162 Env-based chimeric vector expressing V3 epitopes from Env of different clades (Krachmarov et al., 2006). However, neither of these systems can be used to accommodate the cloning of near full sequence primary gp120 antigens as is possible in the system described in the current report.
The availability of this Env-expression system should be a useful tool to produce more pseudotyped viruses that cover a broader spectrum of primary gp120 antigens. The system reported here was mainly developed for the purpose of studying neutralizing antibodies against gp120 but it does not ignore the importance of Nab targeted at gp41 region. Using a common gp41, such as that from SF162, will still allow for the study of neutralizing activities against gp41, such as those similar to mAb, 4E10, and 2F5; however, naturally occurring neutralizing antibodies against gp41 are very rare (Verkoczy et al., 2009
) and more importantly, there are fewer mutations in the gp41 region compared to the gp120 region within each clade. For studies where the goal is to characterize gp41, a gp160 pseudotyped virus can be produced using the full length Env sequences, as described previously (Li et al., 2005
; Montefiori, 2004
The data in the current report also demonstrated that heterologous gp120 and gp41 pairs can be used to produce functional Env as part of a pseudotyped virus. Based on the analysis of HIV-1 gp120 gene sequences available in database, the two restriction sites used to produce SF162-Z were very rare, thus potentially allowing many primary gp120 antigens to take advantage of the system reported here. However, only two examples from clade B and clade C were tested in this pilot study. Further observation is needed once more chimeric Env antigens are produced by using this system to understand if gp120 antigens from additional clades can also be used in this system.
In summary, this new SF162-Z vector should offer a convenient tool for the production of more pseudotyped viruses, particularly for gp120 antigens identified from many field studies. Information obtained from using this technique will provide more information on neutralizing antibody responses during natural infection, which can guide further the development of effective vaccines against HIV-1.