Vaccine escape recombinants emerge after pneumococcal vaccination in the United States

Abstract

The heptavalent pneumococcal conjugate vaccine (PCV7) was introduced in the United States (US) in 2000 and has significantly reduced invasive pneumococcal disease; however, the incidence of nonvaccine serotype invasive disease, particularly due to serotype 19A, has increased. The serotype 19A increase can be explained in part by expansion of a genotype that has been circulating in the US prior to vaccine implementation (and other countries since at least 1990), but also by the emergence of a novel "vaccine escape recombinant" pneumococcal strain. This strain has a genotype that previously was only associated with vaccine serotype 4, but now expresses a nonvaccine serotype 19A capsule. Based on prior evidence for capsular switching by recombination at the capsular locus, the genetic event that resulted in this novel serotype/genotype combination might be identifiable from the DNA sequence of individual pneumococcal strains. Therefore, the aim of this study was to characterise the putative recombinational event(s) at the capsular locus that resulted in the change from a vaccine to a nonvaccine capsular type. Sequencing the capsular locus flanking regions of 51 vaccine escape (progeny), recipient, and putative donor pneumococci revealed a 39 kb recombinational fragment, which included the capsular locus, flanking regions, and two adjacent penicillin-binding proteins, and thus resulted in a capsular switch and penicillin nonsusceptibility in a single genetic event. Since 2003, 37 such vaccine escape strains have been detected, some of which had evolved further. Furthermore, two new types of serotype 19A vaccine escape strains emerged in 2005. To our knowledge, this is the first time a single recombinational event has been documented in vivo that resulted in both a change of serotype and penicillin nonsusceptibility. Vaccine escape by genetic recombination at the capsular locus has the potential to reduce PCV7 effectiveness in the longer term.

Figure 1. Schematic of the Pneumococcal Genome

Housekeeping genes characterised in the MLST scheme (aroE, gdh, gki, recP, spi, xpt, and ddl), the genes for PBPs (pbp2x, -1a, -3, -2b, -2a, and -1b), the capsular locus flanked by the dexB and aliA genes, and the region around the capsular locus that was sequenced in this study (shown also in expanded version) are depicted. The capsular loci of serotypes 4 and 19A are 20.9 kb and 18.6 kb, respectively, in length.

Figure 2. Sequencing Results among Progeny, Donor, and Recipient Pneumococci, including Differences in pbp2x and pbp1a Genes

P₁ and P_1variants, and the P₂ and P_2variant, are each represented by one P₁ and P₂ schematic, respectively, for simplicity. Unique regions of sequence are indicated by variations of shading and patterns, e.g. D₁, P₁, D₄, P₂ all have the same pbp2x sequence (black), whereas R₁ has a different pbp2x sequence (speckled). (A) Schematic of the recombinational crossover points revealed in the major group of progeny strains, ST695^19A. Three SNPs marked and confirmed the upstream crossover region and two SNPs marked and confirmed the downstream crossover region in the progeny strains, as shown with black arrows. The length of the recombinational fragment was 38.6 kb (10.3 kb upstream + 18.6 kb capsular locus + 9.7 kb downstream). (B) Illustration of the second recombinational event. ST2365^19A is identical to ST645^19A in the entire sequenced region; no obvious crossover points have yet been revealed and the recipient is unknown.

Figure 3. eBURST Analysis of Closely Related STs Found in This Study and in the MLST Database (http://www.mlst.net/) as of March 2007

STs described in the current study are indicated with arrows and boxes. The size of each circle is proportional to the number of isolates of that ST, the blue circle indicates the founder, ST247, of the entire clonal complex, and the yellow circles indicate subgroup founders (STs that have at least two single-locus variants). Strains from the ST247 founder complex have been recovered throughout Europe, South America, and the US (ST2365 recovered only in the US), from 1978 to 2005. The allelic profile of ST247 is aroE 16, gdh 13, gki 4, recP 5, spi 6, xpt 10, and ddl 14. Strains from the ST246 subgroup have been detected in Spain, Australia, and the United Kingdom from 1997 to 2005; strains from the ST899 subgroup have been recovered in the US and the Czech Republic from 1999 to 2002; and strains of the ST695 subgroup have been detected in the US and South Africa from 1999 to 2005. Pneumococcal strains of all STs in this diagram are serotype 4 except where indicated; however, the serotype 19A strains have only been reported in the US to date, and only since 2003. P₁, P_1var6, P₂, and P₃ refer to the type of progeny strains (see Table 1).

Figure 4. Sequence Alignments of pbp2x and pbp1a (2,253 and 2,160 Base Pairs, Respectively, in Total Length), Showing the Polymorphic Sites (Numbered Vertically) in Each of the PBP Alleles Present in This Dataset

Note that pbp1a is reverse complemented. Allele 1 for each gene corresponds to the penicillin-susceptible TIGR4 pneumococcal strain [38]. Pbp1a_allele 5 and pbp1a_allele 5rec are identical apart from base pairs 1784 and 2009, which mark the downstream recombinational crossover point in progeny strains ST695^19A.