Genome and methylome evolution of H. pylori during acute infection

6.1.1. The mutation rates during early-stage of infection are in agreement with the mutation rates in chronic infections

In manuscript I, we analyzed whole-genome sequences of isolates from 12 human volunteers who were given a prophylactic vaccine candidate or placebo and subsequently challenged with a fully virulent H. pylori strain (BCM-300). Afterwards, the volunteers were treated with antibiotics to eradicate the infection (Malfertheiner, Selgrad et al. 2018). In vivo genome evolution of H. pylori and the calculation of the mutation rates from sequential and paired isolates from chronic infections have been studied extensively (Morelli, Didelot et al. 2010, Kennemann, Didelot et al. 2011, Didelot, Nell et al. 2013). The estimated mutation rates during long-term infections were found to be higher than for most of the bacteria analyzed so far. Our whole-genome comparisons of the re-isolates revealed that mutation rates during early-stage (i.e. the first 10-12 weeks) infection were in agreement with those calculated for strains from chronically infected individuals. We did not observe any recombination event, which is plausible, because the volunteers were H. pylori-negative until they were challenged

Discussion

120 with the BCM-300 strain. In a previous study from our group, two isolates from volunteers who participated in a vaccination trial and were challenged with a cagPAI-negative strain called BCS 100 (Aebischer, Bumann et al. 2008) were sequenced using 454 sequencing technology (Kennemann, Didelot et al. 2011). In an ongoing study (Estibariz, Suerbaum et al., unpublished data) we sequenced antrum and corpus isolates that were harvested 10 weeks post-infection from volunteers who participated in the same vaccination trial (Aebischer, Bumann et al. 2008). We calculated the average mutation rate as 4.50 × 10⁻⁶ mutations per site per year, which was also in agreement with previous estimates of the mutation rate during chronic infection. In contrast to our results, in the only available other study investigating the genetic evolution of H. pylori during acute infection, Linz and colleagues (Linz, Windsor et al. 2014) reported a mutation rate 140-fold higher than our estimates, and a high recombination rate. Frequent exchange of DNA usually occurs during mixed infections (Falush, Kraft et al. 2001). Thus, it is likely that in the study conducted by Linz and colleagues there was an ongoing mixed infection. In this particular study, two initially H. pylori positive human volunteers were re-infected with H. pylori after having received eradication therapy with antibiotics. The success of the eradication therapy was monitored only by UBTs. In our samples, although some UBTs were negative for H. pylori infection, we were able to culture bacteria from gastric biopsies. Therefore, in the context of challenge trials, negative UBTs do not exclude the presence of low levels of H. pylori infection. It is possible that in the study conducted by Linz et al. (Linz, Windsor et al. 2014), the infection was not fully eradicated by the antibiotic treatment and, therefore, the strains analyzed could be part of the preexisting infection. Our results showed no evidence of recombination in the absence of a second unrelated H. pylori strain.

In conclusion, the challenge of H. pylori-negative human volunteers with a reference H. pylori strain in the context of a carefully monitored clinical study, and the subsequent analysis of H. pylori isolates directly evolved from the challenge strain permits an accurate investigation of mutation rates during early-stage infection (Kennemann, Didelot et al. 2011, Nell, Estibariz et al. 2018). The major limitations in these studies (manuscript I and unpublished data) are the small number of isolates per individual that we obtained. Moreover, this type of infection studies do not allow the investigation of recombination events due to the absence of mixed infections.

6.1.2. Variation of OMP-related genes and virulence factors during acute infection

Recombination and mutations during chronic infection generate allelic variation in H. pylori, which is thought to be important in the adaptation to selective pressures encountered in novel stomach niches after transmission to a new host (Suerbaum and Josenhans 2007). In manuscript I, we observed that several isolates displayed sequence changes within OMP-encoding genes. The same phenomenon was previously reported for chronic infection studies. Several isolates carried mutations within genes of

121 the hop/hof/hor families or switched the activity of the adhesins sabA and sabB due to phase-variation and intra-chromosomal recombination events in these two adhesins. These observations were in agreement with the results of genome analyses of H. pylori isolates from chronically infected individuals that demonstrated that OMP-related genes show a significantly higher tendency to have genetic changes during in vivo colonization (Kennemann, Didelot et al. 2011, Krebes, Didelot et al.

2014). In an ongoing study (Estibariz, Suerbaum et al., unpublished data); we observed that several isolates contained mutations in babA. It was previously shown that there is strong selective pressure affecting the major adhesin BabA during the colonization of humans (Colbeck, Hansen et al. 2006, Nell, Kennemann et al. 2014), Rhesus monkeys (Solnick, Hansen et al. 2004) and rodents (Styer, Hansen et al. 2010).

Modifications in OMP-related genes seem to occur in many bacteria to establish the infection. For example, genetic diversification of adhesins and OMP-related genes was observed in Salmonella enterica serovar Typhimurium (Yue, Han et al. 2015) or in Burkholderia dolosa (Lieberman, Flett et al.

2014). Quick diversification of OMP-associated genes might be important for H. pylori in the adaptation to new stomach niches, or to new host individuals. A recent study from our group has provided support for this hypothesis: the study analyzed gastric biopsies from three stomach regions of 16 H. pylori-infected individuals. The results showed that there is an association between gene polymorphisms affecting motility, chemotaxis and OMPs, and the adaptation to different stomach parts (antral and oxyntic mucosa) (Ailloud, Didelot et al. 2019).

In manuscript I, we also observed changes in two major virulence factors of H. pylori, the cagPAI and VacA. Three of the 12 re-isolates lost cagPAI function due to frameshift mutations in cagY, or the insertion of a mobile element in cagE. In addition, two isolates containing non-synonymous SNPs in cagA and cagW showed a reduction in IL-8 induction and another isolate carried additional cagA copies. Thus, at least in the context of the strain BCM-300, we observed a selection against cagPAI function in multiple individuals. It was reported before that the cagPAI could be partially or completely lost during chronic infections (Bjorkholm, Lundin et al. 2001, Kraft, Stack et al. 2006, Ailloud, Didelot et al. 2019). Modifications in the cagPAI and its ability to induce IL-8 may have an impact on adaptation to new hosts by modulating the inflammatory response of the gastric mucosa.

The ability to produce VacA was abrogated in three isolates from the vaccine group due to stop codons in the gene sequence. Although diverse allelic variants of vacA displaying different toxicities and the loss of vacA activity have been already identified in vivo (Falush, Kraft et al. 2001, Aviles-Jimenez, Letley et al. 2004), VacA inactivation in these three isolates from the vaccination group possibly occurred as a response to vaccine-induced selective pressure. Interestingly, VacA has been reported to be an important factor in H. pylori colonization of animal models, although it was not completely required

Discussion

122 for colonization (Wirth, Beins et al. 1998, Salama, Otto et al. 2001, Winter, Letley et al. 2014). Thus, inactivation of VacA activity could affect H. pylori colonization, but it might also be a way to evade the immune-induced response caused by the vaccine.

The study reported in manuscript I, is one of the very first studies (Kennemann, Didelot et al. 2011, Linz, Windsor et al. 2014) investigating genome adaptation of H. pylori during early-stage infection.

We showed that genetic changes affecting OMP-related genes and virulence factors occurred early in H. pylori infection, potentially contributing to the rapid adaptation of this pathogen to a novel gastric niche. Thus, early modulation of adhesion and virulence factor activity might maintain a balance between the pathogenicity of the bacteria and the immune response of the host, favoring H. pylori to establish a chronic infection.

6.1.3. Vaccine-induced modulation of virulence factors

The rapid increase of bacterial resistance to antibiotics is a major concern. There is a need for developing novel approaches to stop bacterial infections. Vaccines against H. pylori could end the transmission and produce a decrease in antibiotic resistance since fewer antimicrobials would be prescribed. For example, a vaccine against Streptococcus pneumoniae was shown to be effective in immunocompetent patients and reduced the rate of pneumococcal infections (Shapiro, Berg et al.

1991, Daniels, Rogers et al. 2016).

In manuscript I, H. pylori isolates were obtained from human volunteers that were given a prophylactic vaccine candidate or placebo. The vaccine was composed of three recombinant H. pylori antigens (VacA, CagA, and NAP, a neutrophil-activating protein)(Malfertheiner, Selgrad et al. 2018). Although well tolerated and capable of inducing an immune response in the volunteers, the vaccine was ineffective against H. pylori infection. Our whole-genome analysis showed that three of the seven isolates from the vaccine group displayed premature stop codons in vacA. In contrast, the analysis of whole genomes of isolates from another vaccination and H. pylori challenge study (Aebischer, Bumann et al. 2008) did not show modifications within these major virulence factors (Estibariz, Suerbaum et al., unpublished data). The vaccine used in this study was a Salmonella Typhi Ty21a strain expressing H. pylori urease. Thus, despite the small cohort available to us the use of VacA in the prophylactic vaccine (manuscript I) likely led to a selection of isolates with a disrupted vacA gene to avoid the immune response.

In both vaccine studies (manuscript I and Estibariz, Suerbaum et al., unpublished data), we showed that OMP-related genes and LPS were prone to modifications, suggesting that these genetic changes might help H. pylori escape the immune system. Immune system avoidance due to vaccine-related selective pressure has been reported in other microorganisms. For example, despite the effectivity of the Hib vaccine preventing Haemophilus influenzae infections, the wide usage of the vaccine caused

123 the emergence of capsular polysaccharide variants, one of the main virulence factors of this bacterium.

Modifications within this capsular polysaccharide resulted in non-typeable pathogenic H. influenzae serotypes (Agrawal and Murphy 2011).

The development of a successful vaccine is challenging due to the great genetic diversity between H.

pylori strains, which are able to adapt quickly to novel niches. Although a vaccine would be very beneficial to stop the transmission, especially in countries where the infection rate is very high with a higher incidence of gastric malignancies, it seems that there are no current efforts by the pharmaceutical industry to develop novel vaccines against H. pylori. A recent review by Sutton & Boag summarizes the status of the H. pylori vaccine development programs from the past years (Sutton and Boag 2018). Most of the studies stopped after preclinical or phase I stages with the exception of one vaccine candidate that reached phase III. The recombinant vaccine was given to children and followed-up to 1 and 3 years. The authors observed a reduced H. pylori infection rate (Zeng, Mao et al. 2015, Sutton and Boag 2018). Despite this reduction, the results should be confirmed after longer periods.

In conclusion, the rapid genetic evolution of H. pylori likely contributes to the ability of the bacteria to escape from the action of the vaccines tested so far. However, the exact mechanisms leading to vaccine failure have not been elucidated. Thus, to develop a successful vaccine candidate, more efforts are needed to understand how H. pylori genetic variability contributes to the avoidance and modulation of the immune system.