Longitudinal analysis of early HIV-1-specific neutralizing activity in an elite neutralizer and in five patients who developed cross-reactive neutralizing activity

Abstract

We previously established that at 3 years postseroconversion, ~30% of HIV-infected individuals have cross-reactive neutralizing activity (CrNA) in their sera. Here we studied the kinetics with which CrNA develops and how these relate to the development of autologous neutralizing activity as well as viral escape and diversification. For this purpose, sera from five individuals with CrNA and one elite neutralizer that were obtained at three monthly intervals in the first year after seroconversion and at multiple intervals over the disease course were tested for neutralizing activity against an established multiclade panel of six viruses. The same serum samples, as well as sera from three individuals who lacked CrNA, were tested for their neutralizing activities against autologous clonal HIV-1 variants from multiple time points covering the disease course from seroconversion onward. The elite neutralizer already had CrNA at 9.8 months postseroconversion, in contrast with the findings for the other five patients, in whom CrNA was first detected at 20 to 35 months postseroconversion and peaked around 35 months postseroconversion. In all patients, CrNA coincided with neutralizing activity against autologous viruses that were isolated <12 months postseroconversion, while viruses from later time points had already escaped autologous neutralizing activity. Also, the peak in gp160 sequence diversity coincided with the peak of CrNA titers. Individuals who lacked CrNA had lower peak autologous neutralizing titers, viral escape, and sequence diversity than individuals with CrNA. A better understanding of the underlying factors that determine the presence of CrNA or even an elite neutralizer phenotype may aid in the design of an HIV-1 vaccine.

Fig 1

Development of cross-reactive neutralizing activity over the course of HIV-1 infection. (A) Breadth and potency of neutralizing activity in longitudinally obtained serum samples from six individuals who had cross-reactive neutralizing activity (CrNA) and three individuals who lacked CrNA in serum at 35 months postseroconversion (post-SC). Serum samples were obtained at different time points post-SC (rightmost column) from one elite neutralizer (C1) and five individuals with CrNA (C2 to C6) and at one time point post-SC from individuals who lacked CrNA (N1 to N3). The virus panel (top) included 6 viruses from subtypes A, B, C, and CRF_AG. Amphotropic murine leukemia virus (aMLV) served as a negative control. Values represent the reciprocal serum dilution at which 50% inhibition of virus infection was achieved. IC₅₀ titers are indicated as follows: −, <1:40; white background, ≥3 times the value for aMLV; light gray background, ≥1:100; dark gray background, ≥1:300; black background, ≥1:1,000. Months post-SC are color coded in the same way based on the geometric mean IC₅₀ across the 6 viruses. (B) Geometric mean IC₅₀s for each patient across the 6 viruses over the course of the infection. The time point at which CrNA was reached is given in parentheses to the right of the patient ID. To fulfill the definition of CrNA, sera had to neutralize ≥4 viruses above background with a geometric mean IC₅₀ of ≥1:97 (dotted line) as indicated on the y axis.

Fig 2

Cross-reactive and autologous neutralizing activity developed with similar kinetics. The height of individual bars indicates the average IC₅₀s in the serum (y axis) at the indicated month post-SC (x axis), as determined by linear regression of IC₅₀s achieved on ≤5 autologous virus variants per time point. C1 to C5, individuals with CrNA; N1 to N3, individuals who lacked CrNA. The geometric mean IC₅₀s across the heterologous 6-virus panel are shown as dashed lines for individuals with CrNA. Patient C6 is absent from this analysis, because no viruses could be isolated.

Fig 3

Longitudinal analysis of changes in the length of gp160 and the number of PNGS. Data for individuals with CrNA (C1 to C5) are grouped at the top, and data for individuals lacking CrNA (N1 to N3) are grouped at the bottom. Each dot represents one clonal virus variant. The horizontal bars indicate mean values per time point. P values above graphs were calculated by ANOVA across all time points, and individual differences were calculated using Bonferroni's multiple-comparison test for independent samples. Shaded areas indicate the presence of CrNA. The lengths of gp160 and V1V2 and the numbers of PNGS in gp160 and V1V2 are shown for both groups. *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s, nonsignificant. Patient C6 is absent from this analysis because no viruses could be isolated.

Fig 4

Diversity and divergence of HIV-1 gp160 sequences. (A) Sequence diversity across all clones of gp160 per time point is shown for individuals with (black lines) and without (gray dashed lines) CrNA, including one elite neutralizer (black dotted line) (C1), following seroconversion. (B) Divergence between subsequent time points during follow-up. Each dot depicts the latest of the subsequent time points for the same individuals for whom results are shown in panel A. (C) The mean diversity across all viral clones isolated within 5 years postseroconversion per individual is shown for individuals with and without CrNA. The elite neutralizer is represented by the gray dot. (D) Sequence diversity (gp160) (right y axis) and geometric mean IC₅₀s across the 6-virus panel (left y axis) per time point depicted across the period of infection for individuals with CrNA. Patient C6 is absent from this analysis because no viruses could be isolated.