To the Editors:
A major obstacle to an HIV cure is a persistent subset of quiescent, infected cells, known as the latent reservoir.1–3 Proviruses in their quiescent state remain undetected by the immune system and impervious to antiretroviral therapy (ART).4,5 On ART interruption, these proviruses can quickly resume viral replication.6–9 One unmet need is a means to safely and effectively unmask these infected cells in the setting of ART. Multiple latency-reversing agents have been investigated for this purpose, but none has yet demonstrated the ability to significantly reduce the size of the latent reservoir, and most have significant safety concerns.10–14
We recently reported that clinical vaccines administered to people living with HIV can induce cellular HIV RNA expression during virally suppressive ART.15 In that study, vaccination was associated with increased immune activation and enhanced HIV-specific responses. However, it remains unknown how vaccine-specific immune responses correlate with HIV activation and whether standard vaccines induce HIV expression selectively from a small pool of antigen-specific, activated HIV-infected cells, or nonselectively, that is, from a broad pool of bystander-activated HIV-infected cells.
Here, we used deep sequencing to characterize HIV reactivation after a standard influenza vaccination in a group of 7 people living with HIV who were virally suppressed with ART.
Methods available as supplemental Digital Content, https://links.lww.com/QAI/B202.
Seven participants who had a median duration of HIV infection of 21 years [interquartile range (IQR): 17–31] were included. Their median age was 59 years (IQR: 55–64). All started ART during chronic infection and achieved sustained viral suppression below 20 copies per milliliter for at least the past 6 months, and were virally suppressed at the time of vaccine administration. Their median CD4+ T-cell count was 613 cells/mm3 (IQR = 327–721). Population characteristics are summarized in Table S2, Supplemental Digital Content, https://links.lww.com/QAI/B202. On enrollment, participants provided a baseline blood sample and then received a 0.5-mL intramuscular injection of a standard influenza vaccine (Fluarix; GSK, La Jolla, CA). Blood samples were collected at baseline and days 2, 4, 7, 14, and 28 after vaccination.
HIV RNA Transcription and Immune Activation After Vaccination
We first measured cell-associated HIV RNA encoding for gag at each sampled time point, in duplicate by digital droplet polymerase chain reaction, to determine which showed increased HIV transcription after vaccination. Participants K2, K3, and K4 had increased copy numbers of gag RNA (median increase 256 copies/106 cells, range: 177–924) in the week after vaccination, relative to their baseline measures; there was a median of 852 copies of HIV gag DNA (range: 143–5769) and 590 copies of HIV gag RNA (range: 79–3481) per 106 cells in these participants (Figures S1a, b, Supplemental Digital Content, https://links.lww.com/QAI/B202).
We next analyzed HLA-DR and CD38 expression (markers of cellular activation) by flow cytometry and found that CD4+ T cells from 3 of the 7 participants (K3, K4, and K5) showed increasing percentages of HLA-DR and CD38 expression during the 4 weeks after vaccination compared with baseline (Figure S1c, Supplemental Digital Content, https://links.lww.com/QAI/B202). We also compared the specificity of immune responsiveness of the participants by measuring influenza-specific immunoglobulin G (IgG) in the serum at baseline and at days 7, 14, and 28. Participants K3, K4, and K7 exhibited the greatest increases in influenza-specific IgG (Figure S1d, Supplemental Digital Content, https://links.lww.com/QAI/B202).
Source of HIV Transcription
To better understand the source of detectable cell-associated HIV RNA, we deeply sequenced HIV DNA and RNA populations in blood from participants K2, K3, and K4 at all collected time points. Deep sequencing of the HIV gag p24, pol reverse transcriptase, and env C2-V3 coding regions was performed. After quality filtering, over 50 million reads were analyzed with a median of 320,452 reads/region/sample (IQR: 198,053–1,494,661). A median of 6 (IQR: 3–11) and 14 (IQR: 8–23) haplotypes per HIV RNA and DNA sample was generated and analyzed. Comparing sequence diversity within the 3 coding regions through pairwise Tamura-Nei 93 distances between reads with at least 100 overlapping base pairs16 showed no significant changes in sequence diversity in any sequenced HIV coding region between prevaccination samples and samples collected 1 month after vaccination (Figure S2, Supplemental Digital Content, https://links.lww.com/QAI/B202). We next assessed viral compartmentalization between HIV DNA and RNA sequences for participants K2, K3, and K4 using a distance-based FST test on collapsed and reexpanded haplotypes and a tree-based Slatkin–Maddison test17 (Table S3, Supplemental Digital Content, https://links.lww.com/QAI/B202). We found no evidence of viral compartmentalization between paired HIV DNA and RNA samples using either method. Overall, maximum-likelihood phylogenetic trees of the HIV DNA and RNA sequences and tree topologies exhibited extensive intermingling of sequences between HIV RNA and DNA populations at each time point (Fig. 1), especially compared with those from D0 and D28 in the other 4 participants (Figure S3, Supplemental Digital Content, https://links.lww.com/QAI/B202). Together, these results suggested that the vaccine activated HIV DNA populations for transcription nonselectively.
Many current HIV curative strategies have focused on developing methods that induce expression of the virus from infected cells during ART, so that viral proteins are revealed, and cellular reservoirs can be cleared by the host immune response, while ART prevents new cells from being infected.18,19 Prophylactic vaccination represents a potential means of transiently activating the immune system, and has been associated with increased levels of cell-free HIV RNA after vaccinations for influenza,20–29 pneumococcus,30–32 tetanus,28,33 hepatitis B,34 and cholera.35 Our group recently published data generated from a randomized clinical trial, which showed that standard vaccination can increase HIV transcription comparably with what has been observed with the HDACi, Vorinostat.15
Out of our 7 study participants, K2, K3, and K4 most clearly spiked in their HIV gag RNA expression after vaccination (Figures S1a, b, Supplemental Digital Content, https://links.lww.com/QAI/B202). Such variable responsiveness to standard influenza vaccination is common in people living with HIV.36–39 In these 3 participants, HIV DNA population diversity did not change during the 4 weeks after vaccination, suggesting that sampling of infected circulating cells did not change appreciably after vaccination (Figure S3, Supplemental Digital Content, https://links.lww.com/QAI/B202). Maximum-likelihood phylogenetic reconstructions of HIV gag, pol, and env sequences showed extensive overlap between HIV DNA and RNA sequences at all time points (Fig. 1, Figure S4, Supplemental Digital Content, https://links.lww.com/QAI/B202), suggesting that cell-associated HIV RNA sequences were likely derived from a broad, nonselective pool of cellular reservoirs of HIV DNA. This is consistent with a study of the HDACi Panobinostat, which showed panmixis of sequences generated from HIV DNA and RNA populations in circulating peripheral blood mononuclear cell.40
Our study has several limitations. First, our sample size cannot allow for broad generalization to others who have HIV reactivation after influenza vaccination. Because this was a pilot study and not a clinical trial, some people may have selective activation of HIV reservoirs after vaccination. Along these lines, increased cell-associated HIV RNA after vaccination may be due to factors other than vaccination. To explore this further, we chose the 3 participants with the best evidence for increased HIV expression after vaccination (Figures S1a, b, Supplemental Digital Content, https://links.lww.com/QAI/B202). In our previous randomized clinical trial of a larger cohort of people receiving a schedule of vaccines, we were able to detect significant increases in copy numbers of cell-associated, unspliced gag after vaccination.15 Next, our number of sequence reads suggests high levels of polymerase chain reaction amplification, which can introduce primer biases. However, if template selection based on nonideal annealing temperatures occurred, we would expect our data to underestimate the true number of haplotypes and bias in amplification. An underestimation or biased amplification of templates between HIV RNA and DNA populations would likely increase the observation of compartmentalization and thus selective activation, which we did not detect. Along these lines, we did not barcode HIV templates before amplification. Although this decreased amplification bias, it precluded us from measuring clonal expansion of HIV DNA or large bursts of HIV RNA from a single provirus in our samples; however, such conditions would be expected to also increase the potential for observation of compartmentalization, which we did not detect. Finally, our compartmentalization analyses may have been confounded by the number of HIV DNA and RNA haplotypes analyzed. To reduce the likelihood of sampling bias, we grouped all time points for each participant. Compartmentalization was detected in some samples when we analyzed each time point individually (data not shown), but this was likely due to limited HIV RNA haplotypes available for analysis at each time point.
The hunt is on for strategies to provoke HIV expression from quiescent cellular reservoirs and then eradicate the exposed, infected cells. Earlier studies examining the effects of clinical vaccinations on people living with HIV suggested that vaccines can potently activate cellular reservoirs of HIV. Here, we demonstrated that HIV RNA was broadly expressed from a phylogenetically representative pool of circulating cellular reservoirs after vaccination. The ability to reactivate a diverse pool of cellular HIV reservoirs will be critical to HIV-eradication approaches. Our findings are a proof of concept that standard clinical vaccines can broadly reactivate latent HIV from reservoirs suppressed with ART. Standard influenza vaccination will not cure anyone of HIV. However—after further investigation to determine the mechanisms of HIV activation and improve their potency—vaccinations could become a relatively safe addition to various cure efforts.
1. Richman DD, Margolis DM, Delaney M, et al. The challenge of finding a cure for HIV infection. Science. 2009;323:1304–1307.
2. Trono D, Van Lint C, Rouzioux C, et al. HIV persistence and the prospect of long-term drug-free remissions for HIV-infected individuals. Science. 2010;329:174–180.
3. Finzi D, Hermankova M, Pierson T, et al. Identification of a reservoir for HIV-1 in patients on highly active antiretroviral therapy. Science. 1997;278:1295–1300.
4. Chomont N, El-Far M, Ancuta P, et al. HIV reservoir size and persistence are driven by T cell survival and homeostatic proliferation. Nat Med. 2009;15:893–900.
5. Gandhi RT, Bosch RJ, Aga E, et al. No evidence for decay of the latent reservoir in HIV-1-infected patients receiving intensive enfuvirtide-containing antiretroviral therapy. J Infect Dis. 2010;201:293–296.
6. Ruiz L, Martinez-Picado J, Romeu J, et al. Structured treatment interruption in chronically HIV-1 infected patients after long-term viral suppression. AIDS. 2000;14:397–403.
7. Papasavvas E, Ortiz GM, Gross R, et al. Enhancement of human immunodeficiency virus type 1-specific CD4 and CD8 T cell responses in chronically infected persons after temporary treatment interruption. J Infect Dis. 2000;182:766–775.
8. Orenstein JM, Bhat N, Yoder C, et al. Rapid activation of lymph nodes and mononuclear cell HIV expression upon interrupting highly active antiretroviral therapy in patients after prolonged viral suppression. AIDS. 2000;14:1709–1715.
9. Wong JK, Hezareh M, Gunthard HF, et al. Recovery of replication-competent HIV despite prolonged suppression of plasma viremia. Science. 1997;278:1291–1295.
10. Sung JA, Pickeral J, Liu L, et al. Dual-Affinity Re-Targeting proteins direct T cell-mediated cytolysis of latently HIV-infected cells. J Clin Invest. 2015;125:4077–4090.
11. Tsai P, Wu G, Baker CE, et al. In vivo analysis of the effect of panobinostat on cell-associated HIV RNA and DNA levels and latent HIV infection. Retrovirology. 2016;13:36.
12. Banga R, Procopio FA, Cavassini M, et al. In vitro reactivation of replication-competent and infectious HIV-1 by histone deacetylase inhibitors. J Virol. 2015;90:1858–1871.
13. Elliott JH, Wightman F, Solomon A, et al. Activation of HIV transcription with short-course vorinostat in HIV-infected patients on suppressive antiretroviral therapy. PLoS Pathog. 2014;10:e1004473.
14. Jiang G, Dandekar S. Targeting NF-kappaB signaling with protein kinase C agonists as an emerging strategy for combating HIV latency. AIDS Res Hum Retroviruses. 2015;31:4–12.
15. Yek C, Gianella S, Plana M, et al. Standard vaccines increase HIV-1 transcription during antiretroviral therapy. AIDS. 2016;30:2289–2298.
16. Tamura K, Nei M. Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Mol Biol Evol. 1993;10:512–526.
17. Slatkin M, Maddison WP. A cladistic measure of gene flow inferred from the phylogenies of alleles. Genetics. 1989;123:603–613.
18. Deeks SG. HIV: shock and kill. Nature. 2012;487:439–440.
19. Margolis DM. How might we cure HIV? Curr Infect Dis Rep. 2014;16:392.
20. Ho DD. HIV-1 viraemia and influenza. Lancet. 1992;339:1549.
21. O'Brien WA, Grovit-Ferbas K, Namazi A, et al. Human immunodeficiency virus-type 1 replication can be increased in peripheral blood of seropositive patients after influenza vaccination. Blood. 1995;86:1082–1089.
22. Staprans SI, Hamilton BL, Follansbee SE, et al. Activation of virus replication after vaccination of HIV-1-infected individuals. J Exp Med. 1995;182:1727–1737.
23. Rosok B, Voltersvik P, Bjerknes R, et al. Dynamics of HIV-1 replication following influenza vaccination of HIV+ individuals. Clin Exp Immunol. 1996;104:203–207.
24. Tasker SA, O'Brien WA, Treanor JJ, et al. Effects of influenza vaccination in HIV-infected adults: a double-blind, placebo-controlled trial. Vaccine. 1998;16:1039–1042.
25. Gunthard HF, Wong JK, Spina CA, et al. Effect of influenza vaccination on viral replication and immune response in persons infected with human immunodeficiency virus receiving potent antiretroviral therapy. J Infect Dis. 2000;181:522–531.
26. Kolber MA, Gabr AH, De La Rosa A, et al. Genotypic analysis of plasma HIV-1 RNA after influenza vaccination of patients with previously undetectable viral loads. AIDS. 2002;16:537–542.
27. Vigano A, Bricalli D, Trabattoni D, et al. Immunization with both T cell-dependent and T cell-independent vaccines augments HIV viral load secondarily to stimulation of tumor necrosis factor alpha. AIDS Res Hum Retroviruses. 1998;14:727–734.
28. Ostrowski MA, Krakauer DC, Li Y, et al. Effect of immune activation on the dynamics of human immunodeficiency virus replication and on the distribution of viral quasispecies. J Virol. 1998;72:7772–7784.
29. Ramilo O, Hicks PJ, Borvak J, et al. T cell activation and human immunodeficiency virus replication after influenza immunization of infected children. Pediatr Infect Dis J. 1996;15:197–203.
30. Calmy A, Bel M, Nguyen A, et al. Strong serological responses and HIV RNA increase following AS03-adjuvanted pandemic immunization in HIV-infected patients. HIV Med. 2012;13:207–218.
31. Brichacek B, Swindells S, Janoff EN, et al. Increased plasma human immunodeficiency virus type 1 burden following antigenic challenge with pneumococcal vaccine. J Infect Dis. 1996;174:1191–1199.
32. Negredo E, Domingo P, Sambeat MA, et al. Effect of pneumococcal vaccine on plasma HIV-1 RNA of stable patients undergoing effective highly active antiretroviral therapy. Eur J Clin Microbiol Infect Dis. 2001;20:287–288.
33. Stanley SK, Ostrowski MA, Justement JS, et al. Effect of immunization with a common recall antigen on viral expression in patients infected with human immunodeficiency virus type 1. N Engl J Med. 1996;334:1222–1230.
34. Rey D, Krantz V, Partisani M, et al. Increasing the number of hepatitis B vaccine injections augments anti-HBs response rate in HIV-infected patients. Effects on HIV-1 viral load. Vaccine. 2000;18:1161–1165.
35. Ortigao-de-Sampaio MB, Shattock RJ, Hayes P, et al. Increase in plasma viral load after oral cholera immunization of HIV-infected subjects. AIDS. 1998;12:F145–F150.
36. Atashili J, Kalilani L, Adimora AA. Efficacy and clinical effectiveness of influenza vaccines in HIV-infected individuals: a meta-analysis. BMC Infect Dis. 2006;6:138.
37. Talbot HK, Griffin MR, Chen Q, et al. Effectiveness of seasonal vaccine in preventing confirmed influenza-associated hospitalizations in community dwelling older adults. J Infect Dis. 2011;203:500–508.
38. Talbot HK, Zhu Y, Chen Q, et al. Effectiveness of influenza vaccine for preventing laboratory-confirmed influenza hospitalizations in adults, 2011–2012 influenza season. Clin Infect Dis. 2013;56:1774–1777.
39. Havers F, Sokolow L, Shay DK, et al. Case-control study of vaccine effectiveness in preventing laboratory-confirmed influenza hospitalizations in older adults, United States, 2010–2011. Clin Infect Dis. 2016;63:1304–1311.
40. Barton K, Hiener B, Winckelmann A, et al. Broad activation of latent HIV-1 in vivo. Nat Commun. 2016;7:12731.