Gag sequence variation in a human immunodeficiency virus type 1 transmission cluster influences viral replication fitness

Three men from a proven homosexual human immunodeficiency virus type 1 (HIV-1) transmission cluster showed large variation in their clinical course of infection. To evaluate the effect of evolution of the same viral variant in these three patients, we analysed sequence variation in the capsid protein and determined the impact of the observed variation on viral replication fitness in vitro. Viral gag sequences from all three patients contained a mutation at position 242, T242N or T242S, which have been associated with lower virus replication in vitro. Interestingly, HIV-1 variants from patients with a progressive clinical course of infection developed compensatory mutations within the capsid that restored viral fitness, instead of reversion of the T242S mutation. In HIV-1 variants from patient 1, an HLA-B57(+) elite controller, no compensatory mutations emerged during follow-u

HIV-1 replication fitness of HLA-B*57/58:01 CTL escape variants is restored by the accumulation of compensatory mutations in Gag

Expression of HLA-B*57 and the closely related HLA-B*58:01 are associated with prolonged survival after HIV-1 infection. However, large differences in disease course are observed among HLA-B*57/58:01 patients. Escape mutations in CTL epitopes restricted by these HLA alleles come at a fitness cost and particularly the T242N mutation in the TW10 CTL epitope in Gag has been demonstrated to decrease the viral replication capacity. Additional mutations within or flanking this CTL epitope can partially restore replication fitness of CTL escape variants. Five HLA-B*57/58:01 progressors and 5 HLA-B*57/58:01 long-term nonprogressors (LTNPs) were followed longitudinally and we studied which compensatory mutations were involved in the restoration of the viral fitness of variants that escaped from HLA-B*57/58:01-restricted CTL pressure. The Sequence Harmony algorithm was used to detect homology in amino acid composition by comparing longitudinal Gag sequences obtained from HIV-1 patients positive and negative for HLA-B*57/58:01 and from HLA-B*57/58:01 progressors and LTNPs. Although virus isolates from HLA-B*57/58:01 individuals contained multiple CTL escape mutations, these escape mutations were not associated with disease progression. In sequences from HLA-B*57/58:01 progressors, 5 additional mutations in Gag were observed: S126N, L215T, H219Q, M228I and N252H. The combination of these mutations restored the replication fitness of CTL escape HIV-1 variants. Furthermore, we observed a positive correlation between the number of escape and compensatory mutations in Gag and the replication fitness of biological HIV-1 variants isolated from HLA-B*57/58:01 patients, suggesting that the replication fitness of HLA-B*57/58:01 escape variants is restored by accumulation of compensatory mutations

ESBL/pAmpC-producing Escherichia coli and Klebsiella pneumoniae carriage among veterinary healthcare workers in the Netherlands.

The prevalence of ESBL-E/K carriage was 9.8% (47/482; 95%CI 7.4-12.7). The most frequently occurring ESBL genes were blaCTX-M-15, blaCTX-M-14 and blaDHA-1. The predominant sequence type was ST131. None of the occupation related factors, such as contact with specific animal species, were significantly associated with ESBL-E/K carriage, whereas travel to Africa, Asia or Latin America in the past 6 months (OR 4.4), and stomach/bowel complaints in the past 4 weeks (OR 2.2) were. Sixteen of 33 initially ESBL-E/K positive participants (48.5%) tested positive again 6 months later, in 14 persons the same ESBL gene and E. coli ST was found. Four of 23 (17.4%) household members carried ESBL-E/K, in three persons this was the same ESBL gene and E. coli ST as in the veterinary healthcare worker

Low level of HIV-1 evolution after transmission from mother to child

Mother-to-child HIV-1 transmission pairs represent a good opportunity to study the dynamics of CTL escape and reversion after transmission in the light of shared and non-shared HLA-alleles. Mothers share half of their HLA alleles with their children, while the other half is inherited from the father and is generally discordant between mother and child. This implies that HIV-1 transmitted from mother to child enters a host environment to which it has already partially adapted. Here, we studied viral evolution and the dynamics of CTL escape mutations and reversion of these mutations after transmission in the context of shared and non-shared HLA alleles in viral variants obtained from five mother-to-child transmission pairs. Only limited HIV-1 evolution was observed in the children after mother-to-child transmission. Viral evolution was mainly driven by forward mutations located inside CTL epitopes restricted by HLA alleles inherited from the father, which may be indicative of CTL pressur

Characteristics of patients positive for HLA-B*57 or HLA-B*58:01 and the Gag sequences analyzed.

<p>L: long term non-progressor; P: progressor. S: date of seroconversion for HIV-1 antibodies during active follow-up in the cohort. E: HIV+ entry into the cohort.</p

Accumulation of mutations in Gag correlates with a higher replication fitness.

<p>Spearman correlation analysis of the total number of escape and compensatory mutations associated with HLA-B*57/58:01 and the maximum p24 value observed for biological HIV-1 variants obtained from HLA-B*57/58:01 progressors and LTNPs in an <i>in vitro</i> replication assay.</p

Sequence variation in Gag affects viral replication fitness.

<p>A. Sequence Harmony analysis detecting amino acid changes associated with the presence of HLA-B*57/58:01, irrespective of disease course. Longitudinal Gag sequences obtained from HLA-B*57/58:01 progressors and LTNPs (n = 10) were compared to Gag sequences obtained from patients negative for HLA-B*57/58:01 (n = 19). An SH value of 0.90 was used as cut-off. Six amino acid changes were identified to be specifically associated with the presence of HLA-B*57/58:01: I147L, V159I, S173T, T242N, G248A and T280V. Amino acid positions in capsid associated with HLA-B*57/58:01 are shown in red. B. Sequence Harmony analysis detecting amino acid changes associated with disease progression in HLA-B*57/58:01 patients. Gag sequences obtained from late in infection of HLA-B*57/58:01 progressors (n = 5) and HLA-B*57/58:01 LTNPs (n = 5) were compared. An SH value of 0.90 was used as cut-off. Five amino acid changes were identified to be associated with HLA-B*57/58:01 disease progression: S126N, L215T, H219Q, M228I and N252H. Amino acid positions in capsid associated with HLA-B*57/58:01 disease progression are shown in red. C. Replication kinetics (days 2–17) of constructed NL4-3.Ba-L viral variants containing mutations associated with HLA-B*57/58:01 (I147L, V159I, S173T, T242N, G248A and T280V; red), in combination with mutation S126N (orange), L215T (yellow), H219Q (purple), M228I (green), L215T and H219Q (grey), or a combination of all 5 compensatory mutations (S126N, L215T, H219Q, M228I and N252H; blue). Mean p24 concentrations (ng/ml) are given. Error bars represent the standard error of the mean. Data from one representative experiment are shown. D. Replication kinetics of NL4-3.Ba-L and constructed NL4-3.Ba-L viral variants containing mutations associated with HLA-B*57/58:01 (I147L, V159I, S173T, T242N, G248A and T280V) in the absence or presence of compensatory mutations. Normalized mean p24 values on day 11 were compared using the unpaired Student's T test. Statistical significance compared to the mutant virus carrying the mutations associated with the presence of HLA-B*57/58:01 are denoted in red, and significance compared to the virus carrying all mutations associated with the presence of HLA-B*57/58:01 and with disease progression is shown in blue. Statistical significance is indicated as follows: * p<0.05, ** p<0.01, *** p<0.0001. Error bars represent 2.5 – 97.5 percentiles. Data from one representative experiment are shown. E. Replication kinetics of NL4-3.Ba-L and constructed NL4-3.Ba-L viral variants containing mutations associated with HLA-B*57/58:01 (I147L, V159I, S173T, T242N, G248A and T280V) in the absence or presence of compensatory mutations described by Brockman <i>et al</i>. Normalized mean p24 values on day 11 were compared using the unpaired Student's T test. Statistical significance compared to the mutant virus carrying the mutations associated with the presence of HLA-B*57/58:01 are denoted in red, and significance compared to the virus carrying all mutations associated with the presence of HLA-B*57/58:01 and with disease progression is shown in blue. Statistical significance is indicated as follows: * p<0.05, ** p<0.01, *** p<0.0001. Error bars represent 2.5–97.5 percentiles. Data from one representative experiment are shown.</p

Amino acid variation in Gag p17 and p24 associated with HLA-B^*57/58:01 disease progression.

<p>*The Sequence Harmony (SH) score represents overlap in amino acid composition between the two groups analyzed (HLA-B*57/58:01 progressors versus HLA-B*57/58:01 LTNPs). An SH value of 0 indicates no overlap (largest possible differences) while a value of 1 indicates complete overlap (no differences).</p><p>**All Z scores represent P values <0.0001 with correction for multiple testing (450 alignment positions).</p><p>***The most frequent amino acids found at a certain position are depicted as “Dominant”, whereas the “Minor” amino acids are present at least 2 times less frequent than the most frequently occurring amino acid at this position. Amino acids are ordered from highest to lowest frequency.</p

Amino acid variation in Gag p17 and p24 specific for HLA-B^*57/58:01.

<p>*The Sequence Harmony (SH) score represents overlap in amino acid composition between the two groups analyzed (HLA-B*57/58:01 versus non-HLA-B*57/58:01). An SH value of 0 indicates no overlap (largest possible differences) while a value of 1 indicates complete overlap (no differences).</p><p>**All Z scores represent P values <0.0001 with correction for multiple testing (450 alignment positions).</p><p>***The most frequent amino acids found at a certain position are depicted as “Dominant”, whereas the “Minor” amino acids are present at least 2 times less frequent than the most frequently occurring amino acid at this position. Amino acids are ordered from highest to lowest frequency.</p