Evidence for positive selection of hepatitis A virus antigenic variants in vaccinated men-having-sex-with men patients: Implications for immunization policies

Abstract

Background: A huge outbreak in the men-having-sex-with-men (MSM) has hit Europe during the years 2016-2018. Outbreak control has been hampered by vaccine shortages in many countries, and to minimize their impact, reduction of antigen doses has been implemented. However, these measures may have consequences on the evolution of hepatitis A virus (HAV), leading to the emergence of antigenic variants. Cases in vaccinated MSM patients have been detected in Barcelona, opening the possibility to study HAV evolution under immune pressure.

Methods: We performed deep-sequencing analysis of ten overlapping fragments covering the complete capsid coding region of HAV. A total of 14578255 reads were obtained and used for the analysis of virus evolution in vaccinated versus non-vaccinated patients. We estimated maximum and minimum mutation frequencies, and Shannon entropy in the quasispecies of each patient. Non-synonymous (NSyn) mutations affecting residues exposed in the capsid surface were located, with respect to epitopes, using the recently described crystal structure of HAV, as an indication of its potential role in escaping to the effect of vaccines.

Findings: HAV evolution at the quasispecies level, in non-vaccinated and vaccinated patients, revealed higher diversity in epitope-coding regions of the vaccinated group. Although amino acid replacements occurring in and around the epitopes were observed in both groups, their abundance was significantly higher in the quasispecies of vaccinated patients, indicating ongoing processes of fixation.

Interpretation: Our data suggest positive selection of antigenic variants in some vaccinated patients, raising concerns for new vaccination polices directed to the MSM group.

Keywords: Deep-sequencing; MSM; Quasispecies; Vaccine escape-mutants.

Fig. 1

Diagram of the fragments used for the deep-sequencing analysis covering, the 5′ non-coding-region (5′NCR), the complete capsid region (VP4, VP2, VP3, and VP1), and a fragment of the 2A region.

Fig. 2

Phylogenetic analysis of HAV strains isolated from patients of the men-having-sex-with-men (MSM) 2016–2018 outbreak from Barcelona. (a) Phylogenetic tree based on a 255-bp fragment (spanning positions 2958–3230) from the VP1/2A protein. (b) Phylogenetic tree based on a sequence of 91-amino acids (spanning positions 251–300 from VP1 and 1–41 from 2A). (c) Phylogenetic tree based on a 1004-bp fragment (spanning positions 2208–3212) including the complete VP1 sequence and a short fragment of 2A. (d) Phylogenetic tree based on a sequence of 334-amino acids (spanning positions 1–300 from VP1 and 1–35 from 2A). Nucleotide dendrograms were constructed using the neighbour-joining method with distance calculation by the Kimura-2-parameter, and performing a bootstrap of 1000 replicates. Amino acid dendograms were constructed using the Neighbour-Joining method with distance calculation using the Poisson correction method. Samples from vaccinated individuals (n = 5) are underlined, samples from HIV-infected patients (n = 6) are indicated with the “†” symbol. Samples further analysed by MiSeq Illumina (n = 13) are indicated with “*”. The three main circulating strains in Europe related to this outbreak, VRD_521_20166, RIVM-HAV16-0907, and V16-258018A are included, and labelled in bold, as well as five representative samples from a previous MSM outbreak in Barcelona from 2008 to 09 (MSM08-09). Additionally, eleven reference strains representative of all subgenotypes (subgenotype IA: AB020565, EU13137, X83302; subgenotype IB: M14707, M59808, DQ646426, subgenotype IC: HQ401240; subgenotype IIA: AY644676; subgenotype IIB: AY644670; subgenotype IIIA: AB279733; subgenotype IIIB: AB425339), including the HM175 strain used in the TWINRIX vaccine (subgenotype IB), were also added. All tested samples were obtained in the period 2016–2017.

Fig. 3

HAV viral loads. (a) Boxplot of the average virus load in sera from the non-vaccinated and vaccinated groups. (b) Boxplot of the average virus load in sera from HIV-non-infected and HIV-infected groups. Bottom and upper limits of the boxes represent the 25th and 75th percentiles, respectively. When present (n > 9), bottom and upper whiskers represent 10th and 90th percentiles, respectively. The median is represented by a solid black line and the average as a dotted red line.

Fig. 4

Informative content (IC) of each amino acid position along the capsid, and analysis of the frequency of the NSyn mutations. (a) The analysis of the informative content was performed using overlapping windows of 10 amino acids sliding every 3 amino acids. IC values (in bits) range from 0 (maximum variability) to 4·32 (minimum variability). Each point (amino acid position) corresponds to the mean IC from all the sequences of a given group. The black line corresponds to the non-vaccinated group and the red line to the vaccinated group. Values have been normalized by the frequency of each substitution and the number of sequences per group. (b) Frequency (%) of the NSyn mutations detected in the non-vaccinated patients (n = 8; black dots) and vaccinated patients (n = 5; red dots); black dots are more common due to the higher n. The horizontal green line represents the 5% frequency; points over this line represent high-frequency mutations.

Fig. 5

Location of the variants affecting residues in and around the antigenic sites of the HAV capsid. (a) Protomers from non-vaccinated patients (M1, M2, M4, M9, M10, M17, M30, and M47). (b) Protomers from vaccinated patients (M5, M6, M7, M46 and M48). Green protein corresponds to VP2, red to VP3 and blue to VP1. Protomers used to locate the replacements were extracted from the recently resolved crystal structure of HAV [18]. Epitopes (yellow residues) were identified either by mAb escape mutants [19,20] (VP1 and VP3) or mAb footprints [21] (VP2 and VP3). Grey residues represent mutations directly affecting the epitopes. Clear blue residues represent mutations located at a distance of 1–2 residues from the epitopes identified in monoclonal antibodies (mAbs) resistant mutants.

Fig. 6

Analysis of NSyn mutations located in and around the main antigenic sites in the non-vaccinated and vaccinated groups. (a) Percent of all NSyn mutations located in or around the antigenic sites distributed in the VP1 immunodominant site (dark blue), the VP1 glycophorin A binding site (red) and the VP2, VP3 putative receptor binding (clear blue). (b) Average of the abundance of haplotypes bearing the NSyn mutations from (a).