It has been estimated that one million to two million people worldwide are infected with HIV-2 (1
). Similarly to HIV-1, HIV-2 causes AIDS, but with lower rates of transmission, CD4+
T-cell decline, and disease progression (2
). Despite similar levels of integrated viral DNA (proviral DNA), the plasma viral RNA burden (viral load) at comparable CD4+
T-cell counts is significantly lower in HIV-2 infections than in HIV-1 infections, suggesting either that HIV-2 is associated with a lower rate of replication or that HIV-2 is more susceptible to immune control (2–8
). However, despite the lower level of chronic immune activation in HIV-2 infection, both viruses elicit several immune responses that may modulate disease progression, e.g., neutralizing antibodies and cytotoxic T lymphocytes (2
The natural history of HIV-2 infection was not known in detail until recently, and cohort data often lack information on the estimated time of HIV-2 infection, precluding assessment of the true rates of HIV-2 disease progression and of the dynamics of CD4+
T-cell change and plasma viral load during infection (9
). In a recent study, we showed that most individuals infected by HIV-2 progress to disease but at a lower rate than for HIV-1 (11
). Moreover, the role of CD4+
T-cell dynamics in HIV-2 infection was shown to be a strong clinical predictor of disease progression. Thus, both HIV-1 disease progression and HIV-2 disease progression are associated with CD4+
T-cell decline and early initial postseroconversion CD4+
T-cell levels (11–15
Both HIV-1 and HIV-2 evolve rapidly due to high mutation rates, high replication rates, and fast generation times (16
). This results in extensive genetic variability both within and between infected individuals. The association between viral evolutionary rate (5
) and disease progression has been studied extensively for HIV-1, and most studies have suggested that these parameters are positively correlated (18
). Much less is known about HIV-2 intrapatient evolution; whereas some researchers have reported that HIV-2 has a lower evolutionary rate than HIV-1, others have reported the opposite (5
). However, no correlation has been found between the virus evolutionary rate and variations in levels of CD4+
T-cells over time in HIV-2 infection (5
). Importantly, those studies were performed on proviral DNA or on RNA obtained from virus propagated in culture, which may not reflect the circulating virus populations. To the best of our knowledge, differences in HIV-2 evolutionary rates between groups with different rates of disease progression have not been investigated.
Here, we aimed to determine whether faster disease progression and slower disease progression are differentiated by rates of decline of and levels of CD4+
T cells in HIV-2 infection, similarly to what has been previously suggested to be the case in HIV-1 infection (12
), and investigated the association between the disease progression rate and the evolutionary dynamics of HIV-2.
The relationship between HIV evolution and disease progression is fundamental to our understanding of HIV immune control and vaccine design. We recently showed an association between CD4+
T-cell level and HIV-2 disease progression rate (11
). Here we defined relatively faster and slower HIV-2 disease progression using these associations and dissected the associations between HIV-2 evolutionary dynamics and disease progression. Studies addressing these associations for HIV-2 infection have been limited (5
). In HIV-2 infection, disease in many patients progresses slowly, but in some the advance is as fast as that in HIV-1 infection (28–30
). The reasons for this marked heterogeneity are currently not known, but mechanisms similar to those of HIV-1 infection may be involved (11
). To address the hypothesis that viral evolution is associated with disease progression in HIV-2 infection also, we first determined stratifications for relatively faster and slower disease progression based on follow-up data from the entire prospective cohort of police officers in Guinea-Bissau. The analysis showed that although CD4+
T-cell level and decline were independently associated with progression to AIDS, the effect size was largest for the CD4% level or the combined CD4% level/decline stratifications. This observation is in line with previous reports showing that the CD4+
T-cell level at corresponding time points after infection may be a better marker for both HIV-1 and HIV-2 disease progression rates than CD4+
T-cell decline (11
). Moreover, only the CD4+
T-cell level or the combined CD4+
T-cell level/decline stratifications were associated with the evolutionary rate of HIV-2. This observation suggests that the postseroconversion CD4+
T-cell level is associated with the rate of disease progression (32
), whereas the rate of CD4+
T-cell decline during chronic infection can be viewed as an additive component influencing progression in combination with the initial CD4+
T-cell levels. The reasons for and mechanisms that determine the variability of CD4+
T-cell decline range from genetic and biological factors to physiological factors (33
). In those with a low postseroconversion level of CD4 cells, progression to AIDS is faster (and the time to AIDS shorter) than among those with a higher postseroconversion level of CD4 cells (11
). It is possible that events that occur during acute infection dictate the initial postseroconversion levels of CD4 T-positive (T+
) cells in HIV-2 infection also (11
). Thus, a broader assessment of disease progression may provide additional understanding of the mechanisms that drive the disease pathogenesis.
Many HIV-2-infected individuals remain nonprogressors with low viral loads during the course of infection, and HIV-2 sequences can be obtained only from individuals with detectable plasma viral loads (35
). Hence, HIV-2-infected individuals without detectable viremia cannot be assessed in studies of HIV-2 evolution in plasma. However, our assay had a detection limit of 12 RNA copies/ml plasma, indicating that even slow progressors with low viral loads (<50 copies/ml plasma) could be detected.
A strong association between HIV-2 evolutionary rate and disease progression was found in all the studied genetic subregions, except for V3. For HIV-1, the flanking V3 region is known to be highly exposed and immunodominant (36
). By contrast, the V3 region of HIV-2 has been suggested to be more highly concealed from the immune system and to be the least entropic and positively selected part of the C2-C3 region (24
). Our findings of lower evolutionary rates and stronger purifying selection in the C2 and V3 regions support previous suggestions that the immune response of HIV-2-infected individuals may be more highly directed to other regions of the HIV-2 envelope (27
Our HIV-2 evolutionary rate estimates are in the range of what has been reported by others, although direct comparisons are difficult due to differences in the env
regions analyzed and to the use of different phylogenetic models (17
). In a previous study of HIV-1 subtype B based on a similar methodological approach, the evolutionary rate of the HIV-1 V1-V3 region was estimated to be approximately twice as high as our estimates for HIV-2 (39
). The uncertainty of how the HIV-2 evolutionary rate compares with the rate of HIV-1 highlights the need for a direct comparison of HIV-1 evolution to HIV-2 evolution in the same population using the same approaches. Previous studies of HIV-2 intrahost evolution have been based on limited numbers of individuals and time points. The generally low viral loads among HIV-2-infected individuals continue to present a large technical challenge and are likely to have contributed to the paucity of intrahost HIV-2 evolutionary studies (6–8
In a stratified analysis, we found that both nonsynonymous and synonymous substitutions accumulated at a higher rate in faster progressors than in slower progressors. This result suggests generally faster replication rates and shorter generation times for virus populations in faster progressors and is in line with previous reports of increased virus replication rates among immunosuppressed individuals infected with HIV-2 (40
). It is possible that increased replication rates can reduce the generation time in intrahost virus populations and can lead to higher rates of neutral evolution. This has also been suggested to explain the association between disease progression and synonymous substitution rates in HIV-1 infection (17
Previous studies have demonstrated that the env
gene is under purifying selective pressure overall in both HIV-1 and HIV-2 infection, with a few irregularly distributed positively selected sites (42
). Comparisons between HIV-1 disease progressor groups have suggested that slow-disease progressors are associated with a higher number of positively selected sites (44
). Similarly to HIV-1 data, we identified a few positively selected sites in the majority of HIV-2 slow progressors. However, the mean dN/dS rate ratios and the proportions of positively selected sites did not differ between slower and faster progressors. Instead, we found that slow HIV-2 disease progression was associated with a higher level of positive selection on a selected number of surface-exposed residues conserved between HIV-2/SIVsm. It is tempting to speculate that slow progressors may elicit a stronger immune response to highly surface-exposed conserved residues, which may in turn impact viral fitness, since such conserved amino acids are likely to have a functional and structural impact on envelope functions (24
). If true, our findings would be consistent with the concept that hosts who mount a stronger immune response against the infecting virus have greater numbers of positively selected sites and progress to AIDS at a lower rate, which is reflected by higher postseroconversion CD4+
T-cell levels (44
). In line with this, Bohl et al. showed that mutations of conserved residues of HIV-2 envelope resulted in poor envelope function (46
In conclusion, our analyses show a strong association between HIV-2 evolutionary rate and disease progression as determined by CD4% levels. Overall negative selection was demonstrated in the analyzed HIV-2 env fragment, with the proportion of positively selected sites in the range of what has been shown for HIV-1. Interestingly, slow disease progression among HIV-2-infected individuals was associated with higher levels of positive selection on residues conserved between HIV-2 and SIVsm, which may indicate generally reduced viral fitness among these viral variants. Our findings provide new insights into the associations between pathogenesis and intrahost evolution of HIV-2. Still, more studies on how the dynamics of disease progression rate is shaped by the molecular evolution of HIV-2 are warranted. Further knowledge of HIV-2 pathogenesis and comparisons between HIV-1 and HIV-2 will be important to reveal fundamental differences in how these two viruses cause immunodeficiency.
We thank all participants in the study. We also thank Aquilina Sambu, Isabel da Costa, Jacqueline Pereira, Siaca Sambu, and Ana Monteiro Watche at the Health Station of the 2a esquadra, Bissau, and Braima Dabo, Carla Pereira, Julieta Pinto Delgado, Leonvengilda Fernandes Mendes, Ana Monteiro, and Inacio Gomes at the National Public Health Laboratory in Bissau. We also thank Mauno Vihinen for assistance with the Chimera software.
The study was supported by the Department for Research Cooperation (SAREC) at the Swedish International Development Agency (Sida) and the Swedish Research Council (no. 350-2012-6628 and 2016-01417 for J.E.; no. 2016-02285 for M.J.; no. 321-2012-3274 for P.M.). J.E. also acknowledges funding from Swedish Society of Medical Research (SA-2016). P.L. acknowledges funding from the European Union Seventh Framework Program (FP7/2007-2013) under grant agreement no. 260864.
Members of the Sweden Guinea-Bissau Cohort Research (SWEGUB CORE) group include Babetida N’Buna, Antonio Biague, Ansu Biai, Cidia Camara, Joakim Esbjörnsson, Marianne Jansson, Sara Karlson, Jacob Lopatko Lindman, Patrik Medstrand, Fredrik Månsson, Hans Norrgren, Angelica A. Palm, Gülsen Özkaya Sahin, and Zacarias Jose da Silva.
P.M. and J.E. contributed equally to this study. A.A.P., J.E., and P.M. interpreted the data and were responsible for the overall study design. M.J., P.M., and H.N. were responsible for the overall project coordination. F.M., H.N., and A.B. were responsible with respect to the medical and organizational concerns associated with the clinical sites with biological samples of the study participants in the cohort. Z.J.D.S. was responsible for analyses of HIV serology and T-cell count determinations at the laboratory in Guinea-Bissau. F.M. and H.N. coordinated the laboratory and clinical work in Guinea-Bissau and performed database entry and cleaning. A.A.P., J.E., P.M., and Z.S. performed the analyses. A.K., P.L., J.E., and P.M. contributed to the statistical analyses. M.J., S.L.R.-J., and P.L. participated in interpretation of the results. A.A.P., J.E., and P.M. wrote the manuscript. All of us read and approved the manuscript.
We declare that we have no competing financial interests.