A surprising hallmark of RhCMV-SIV vaccine-mediated immunity is that protection against transient SIV infection appears to occur in the absence of a detectable anamnestic T cell response. Protection against viral infection in the absence of memory T cell proliferation is not only an unusual outcome but it is highlighted by the authors in primary research publications (see abstract of Hansen, 2013), review articles (Barouch and Picker, Nature Reviews Microbiology 2014) and public presentations (Picker, Barcelona, 2013). However, with the possible exception of exhausted or dysfunctional T cells, the current consensus among members of the immunological community is that memory T cells typically proliferate in response to productive infection since this represents one of the most basic functions of a memory T cell. As noted in the earlier PubPeer comments on the 2013 Nature study by Hansen et al., these results raise the question – why are RhCMV-SIV specific T cells so different and why don’t they proliferate during SIV infection?
There are two potential answers; 1) RhCMV-induced T cell memory is unique in the sense that these cells can reduce or even clear SIV infection in the absence of detectable proliferation, or 2) the lack of a proliferating, anamnestic T cell response may instead be evidence that SIV infection had not occurred. This second option is problematic because RhCMV vaccines do not elicit neutralizing antibody responses and therefore cannot protect against infection per se, but instead can only provide protection after an initial infection has occurred. If the vaccinated animals do not receive an infectious dose of SIV, then the results of vaccine efficacy could be compromised or invalid since there would be no actual infection for the T cells to protect against. The goal here is to attempt to distinguish between these two possibilities.
Cytomegalovirus is known to induce effector memory T cells and both CD4+ and CD8+ T cells have the capacity to proliferate in response to their specific peptide antigen. For example, human CMV-specific effector memory T cells proliferate in vitro in response to HCMV peptide stimulation (Wallace, 2001 and Suni, 2001). Mouse CMV-specific T cells also proliferate rapidly in response to MCMV infection in vivo (Snyder, 2008). Similar to HCMV and MCMV, RhCMV-induced effector memory T cells appear to proliferate in vivo in response to productive reinfection by RhCMV (Hansen, 2010, 2011, and Malouli, 2014). Together, this indicates that the ability for CMV-specific T cells to proliferate in vitro and in vivo following infection is conserved across at least 3 CMVs among 3 different mammalian hosts.
RhCMV-induced effector memory T cells not only respond to RhCMV infection in vivo, but they appear to proliferate in response to other types of infections as well. As noted before on PubPeer, RhCMV-SIV-specific effector memory T cells appear to undergo substantial proliferation in response to booster vaccination/infection with recombinant Adenovirus serotype 5 vectors expressing SIV antigens (Ad5-SIV, Hansen, 2011). This is particularly impressive because unlike SIVmac239 which causes a brief systemic infection/viremia in protected RhCMV-SIV vaccinated animals, the Ad5-SIV vectors used in these studies are unable to replicate or to spread systemically and yet the associated spikes in RhCMV-SIV-induced effector memory CD4+ and CD8+ T cell proliferation are clearly evident (Fig. 2a and Sup Fig. 10, Hansen, 2011). The ability of RhCMV-induced effector memory T cells to proliferate in vivo is not limited to acute or chronic viral infections either since they also appear to proliferate in response to bacterial infections as well. In recent studies aimed at developing a RhCMV-based vaccine against Mycobacterium tuberculosis (TB), RhCMV-TB specific T cells appeared to proliferate in response to experimental TB infection of rhesus macaques (Picker, Barcelona, 2013).
Is it possible to use effector memory T cell proliferation as a biological readout for determining whether or not an initial viral infection has occurred in vivo? The answer appears to be yes – there are at least 2 studies in which RhCMV-specific T cell proliferation was used to distinguish between RhCMV strains that could (or could not) superinfect RhCMV-immune NHP (Hansen, 2010, Malouli, 2014). The Malouli study is the most informative on this topic because the lack of RhCMV-specific memory T cell proliferation was used to prove that RhCMV-immune animals could not be infected by RhCMVdeltaUS2-11. In these studies (Fig. 3), RhCMV-naïve NHP were infected with RhCMVdeltapp65 to induce a baseline level of RhCMV IE-specific T cell memory. At 659 days after this primary infection, the animals were challenged with 10 million PFU of RhCMVdeltaUS2-11gag virus. As noted by the authors, there was no discernable level of virus-specific memory T cell proliferation since the US2-11 deletion results in an attenuated strain of virus that is unable to infect a RhCMV-immune host. To demonstrate that the RhCMV-specific memory T cells could proliferate if a productive RhCMV infection had occurred, they challenged the same animals with a wild-type strain of RhCMV (RhCMV-WTgag) on day 876 and RhCMV-deltapp65ab-rtn on day 1,107. Both of these viruses are capable of infection/superinfection and both of these viruses appeared to elicit anamnestic T cell responses with readily discernable spikes in RhCMV IE-specific effector memory T cell proliferation.
Based on these well-controlled studies, viral infection results in memory T cell proliferation whereas lack of viral infection is accompanied by lack of an anamnestic memory T cell response. By extension, the lack of an anamnestic T cell response among RhCMV-vaccinated animals after SIV challenge would likewise indicate that these animals were not actually infected with SIV. This is compatible with the theory described previously on PubPeer that the RT-PCR primers used for determining SIV infection status may be crossreactive with RhCMV-SIVgag and may not necessarily be indicative of true SIVmac239 infection. In this scenario, spurious SIV+ RT-PCR results occurring in the absence of an anamnestic T cell response in about half of the vaccinated animals would most likely be due to an experimental artifact of the RT-PCR assay and the animals would remain uninfected, especially if they are prematurely removed from the SIV challenge pool. In contrast, the other half of the vaccinated animals eventually receive an infectious dose of SIV and become chronically infected with progressive disease that is associated with high viremia. The lack of an anamnestic T cell response appears to support the theory of a cross-reactive RT-PCR assay detecting RhCMV-SIVgag in the absence of SIVmac239 infection but the key to fully understanding this data will be to verify the sequence of the SIVgag-specific RT-PCR primers and their sequence alignment with the codon-optimized RhCMV-SIVgag insert.
Human CMV-specific T cells proliferate when they encounter viral peptide antigen in vitro:
CD4(+)CD8(dim) T lymphocytes exhibit enhanced cytokine expression, proliferation and cytotoxic activity in response to HCMV and HIV-1 antigens.
Suni MA1, Ghanekar SA, Houck DW, Maecker HT, Wormsley SB, Picker LJ, Moss RB, Maino VC.
Eur J Immunol. 2001 Aug;31(8):2512-20.
Human cytomegalovirus-specific CD8(+) T-cell expansions contain long-lived cells that retain functional capacity in both young and elderly subjects.
Wallace DL1, Masters JE, De Lara CM, Henson SM, Worth A, Zhang Y, Kumar SR, Beverley PC, Akbar AN, Macallan DC.
Immunology. 2011 Jan;132(1):27-38.
Mouse CMV-specific T cells proliferate when they encounter peptide antigen after viral infection in vivo:
Memory inflation during chronic viral infection is maintained by continuous production of short-lived, functional T cells.
Snyder CM1, Cho KS, Bonnett EL, van Dommelen S, Shellam GR, Hill AB.
Immunity. 2008 Oct 17;29(4):650-9.
Rhesus CMV-specific T cells proliferate when they encounter peptide antigen after viral infection in vivo:
Evasion of CD8+ T cells is critical for superinfection by cytomegalovirus.
Hansen SG, Powers CJ, Richards R, Ventura AB, Ford JC, Siess D, Axthelm MK, Nelson JA, Jarvis MA, Picker LJ, Früh K.
Science. 2010 Apr 2;328(5974):102-6.
Profound early control of highly pathogenic SIV by an effector memory T-cell vaccine.
Hansen SG, Ford JC, Lewis MS, Ventura AB, Hughes CM, Coyne-Johnson L, Whizin N, Oswald K, Shoemaker R, Swanson T, Legasse AW, Chiuchiolo MJ, Parks CL, Axthelm MK, Nelson JA, Jarvis MA, Piatak M Jr, Lifson JD, Picker LJ.
Nature. 2011 May 26;473(7348):523-7.
Cytomegalovirus pp65 limits dissemination but is dispensable for persistence.
Malouli D, Hansen SG, Nakayasu ES, Marshall EE, Hughes CM, Ventura AB, Gilbride RM, Lewis MS, Xu G, Kreklywich C, Whizin N, Fischer M, Legasse AW, Viswanathan K, Siess D, Camp DG 2nd, Axthelm MK, Kahl C, DeFilippis VR, Smith RD, Streblow DN, Picker LJ, Früh K.
J Clin Invest. 2014 May;124(5):1928-44.
Rhesus CMV-SIV-specific T cells proliferate in vivo in response to transient viral infection (recombinant adenovirus serotype 5, rAd5-SIV):
Profound early control of highly pathogenic SIV by an effector memory T-cell vaccine.
Hansen SG, Ford JC, Lewis MS, Ventura AB, Hughes CM, Coyne-Johnson L, Whizin N, Oswald K, Shoemaker R, Swanson T, Legasse AW, Chiuchiolo MJ, Parks CL, Axthelm MK, Nelson JA, Jarvis MA, Piatak M Jr, Lifson JD, Picker LJ.
Nature. 2011 May 26;473(7348):523-7.
Rhesus CMV-TB-specific T cells proliferate in vivo in response to chronic bacterial infection (M. tuberculosis):
Pre-clinical Development of Cytomegalovirus Vectors
Louis Picker, presentation at the AIDS vaccine conference Barcelona Spain October 8, 2013
http://www.aidsvaxwebcasts.org/console/player/21652?mediaType=slideVideo&