By expansion of viral stocks, do you mean that you add more cells to the culture every few days? or do you maintain the cell numbers and just refeed with more media. If you just refeed with media, the cell numbers in culture are limited and the cells die/are exhausted when they are continuously producing virus and hence the virus 'expands' till about day 15 and then the titer at later time points decreases. 

To increase the titer, you can produce the first stock of virus in 293T cells with VSV-G glycoprotein. Pseudotyping with VSV-G will increase infectivity of the virus. I presume that you expand stocks in CEM cells or some T cell line. The initial infection could be higher if you use VSV-G pseudotyped virus. Be warned that VSV-G transduces a great variety of cells and you should take appropriate precautions when working with replication-competent HIV, pseudotyped with VSV-G.

Apart from using polybrene and pseudotyping with VSV-G, it is hard to increase the titer in any other way.

Thank you Sanath for your reply and  great information on stock expansion. Here is what I do:

1. I take the initial virus provided to me

2. Then I add PBMCs (from healthy donors, pre-stimulated for 72 h with PHA-P & IL-2 before infection)

3. Then I add RPMI with 10% FBS, 1% PSA, IL-2 

4. Every 5 days, I spin down the cells and collect the supernatant , keep around 1 mL of the vrus containing supernatant and the cells in the 15 mL tube being used for expansion. I then add around 4 mL media (again RPMI with all the above contents except PHA-P)

5. After about 7 days, I top-up the PBMCs (around 10 million PBMCs resuspended in 1 mL of RPMI-IL-2-10%FBS) in the infection tube.

This is how I do it. When I used the HIV-1 obtained after transfecting HEK293 cells, I get low HIV-titer. 

The same goes with other lab adapted HIV-1 strains as well (these HIV-1 strains had been previously expanded using the same technique mentioned above, with expansion going for about 30 days, with supernatant collected every 5 days).

I get good titer for one newly opened vial of HIV-1 strain obtained from NIH (as a part of NIH reagent program). However, the strains which are not expanding now to an expected titer were also obtained from NIH, the only difference being that, they were passaged for several times in PBMCs. The stocks so obtained, when used for expansion later, do not expand well.

Please let me know as to what can be done. I use polybrene as well. Also, please let me know the protocol for the expansion of VSV-G pseudotyped HIV-1 (I am not sure if I will have access to it, or if the pseudotyped virus is available with us at all). I was thinking of Hut78 cell line as well for the expansion (a T cell line), but haven't used so far for expansion.

I prefer to use PBMCs, since at this time point, I just have a preliminary study, and I have to have the virus stocks asap, whcih I'll be using for inhibition assays using PBMCs.

Thanks & Regards,

Siddhesh

If you are looking to increase the concentration of the virus itself, you could spin the virus through a sucrose cushion to obtain a concentrated stock- That increases the titer of the virus, but not the total yield of the virus. 

To pseudotype the virus with VSV-G, you would transfect 293T cells with a VSV-G expression plasmid along with the HIV virus expression plasmid, like pNL4-3. 2 days later collect the supernatant which contains the HIV particles pseudotyped with VSV-G. In effect, you are creating a virus that can infect using the HIV Env and also VSV-G. You usually get better infectivity through the VSV-G glycoprotein. 

To just expand stocks it may be beneficial to expand the stocks in a T cell line rather than PBMCs. You may want to look into the literature as to what cell lines other people have used for this purpose. CEM-SS cells may be appropriate if my memory serves me right.