New paper: Genome-wide association studies reveal the role of polymorphisms affecting factor H binding protein expression in host invasion by Neisseria meningitidis

In this paper, published in October in PLOS Pathogens, we discovered a novel genetic association between life-threatening invasive meningococcal disease (IMD) and bacterial genetic variation in factor H binding protein (fHbp) through two bacterial genome-wide association studies (GWAS), which we validated experimentally. This was a collaboration with the groups of Chris Tang and Martin Maiden, with the work in my group led by Sarah Earle.

fHbp is an important component of meningococcal vaccines that directly interacts with human complement factor H (CFH). Intriguingly, our discovery that bacterial genetic variation in fHbp associates with increased virulence mirrors an earlier discovery that human genetic variation in CFH associates with increased susceptibility to IMD (Nature Genetics 42: 772).

Our experiments showed that the fHbp risk allele increased expression. Interestingly, increased susceptibility to IMD has been previously associated with elevated CFH expression. Therefore over-expression of either fHbp by the bacterium or CFH by the host appears to increase the risk of IMD. Since complement evasion is necessary for pathogenesis, these insights offer new leads for improving treatment.

Key results from the paper:

  • A GWAS for IMD in 261 meningococci from the Czech Republic highlighted a highly polygenic architecture of meningococcal virulence (see Figure), including capsule biosynthesis genes, the meningococcal disease association island and the new signal near the fba and fHbp genes.
  • A replication GWAS for IMD in 1295 meningococcal genomes belonging to strain ST41/44 downloaded from pubMLST.org validated the novel signal of association near fba and fHbp.
  • SHAPE reactivity analyses revealed that IMD-associated variation in the regulatory region of fHbp disrupted the ability of the cell machinery to commence gene expression.
  • Flow cytometry assays of newly constructed genetically engineered strains, in different temperatures and in the presence and absence of human serum, attributed changes in gene expression to a non-synonymous candidate mutation in the fHbp gene.

In this study, our GWAS relied exclusively on publicly available genome sequences and metadata, highlighting the untapped potential of large-scale open source databases like pubMLST.org, and the value of big data for improving our understanding of disease.



University of Oregon outbreak highlights collaboration between public health and clinical care

By Michelle Forman, senior specialist, media, APHL

University of Oregon outbreak highlights collaboration between public health and clinical care | www.APHLblog.org

In mid-January, a University of Oregon student was diagnosed with Neisseria meningitidis serogroup B, a rare but serious disease. Within one month, three additional students were diagnosed with the same disease, one of whom died. “I was the first assistant on that autopsy,” said Patrick F. Luedtke MD, MPH, senior public health officer and medical director of the Lane County Department of Health & Human Services Community & Behavioral Health clinics. (He’s also a past APHL president.) “The bacteria were everywhere. Neisseria meningitidis takes over the body and wins every battle.”

College campuses like the University of Oregon are perfect breeding grounds for meningococcal disease. Young adults ages 16-21 have higher rates than others, and it is transmitted through close or lengthy contact such as living in close quarters or kissing. So, yeah… meningococcal disease can make its way across a college campus if it isn’t stopped quickly. In fact, there were similar outbreaks at Princeton University and at University of California, Santa Barbara in 2013.

Meningococcal disease is rare, but if a person gets it they are likely to become very sick. Once it is suspected, clinical laboratories can do a test to confirm meningococcal disease and doctors can quickly begin antibiotic treatment. (Oftentimes prophylactic antibiotic treatment is given anyone who had close contact with the sick individual.) But even with quick and proper treatment, approximately 20% of people will have long-term disabilities and 10-15% of people die. The best way to prevent severe illness is to prevent illness all together – decrease the number of people who can get meningococcal disease in the first place – with vaccines. Here’s the kicker, though… Kids in the US receive a quadrivalent meningococcal vaccine at age 11. However, that vaccine only protects kids from serogroup A, C, Y or W-135. What about B, the serogroup found at the University of Oregon?

In October 2014, the FDA approved the first ever N. meningitidis serogroup B vaccine for use in people 10-25 years of age as a three-dose series. In January 2015, the FDA approved another N. meningitidis serogroup B vaccine for use in the same age group as a two-dose series. Neither vaccine has been recommended for routine use yet, but it has been recommended for controlling outbreaks like the one at the University of Oregon. In order to implement a massive campaign to vaccinate all 22,000 students, CDC needed to know that there had been at least three confirmed serogroup B cases within a three month period. The clinical test that confirmed meningococcal disease in each of the four patients wasn’t enough, though. Not only are clinical laboratories often without the capabilities to serotype meningococcal disease, the serogroup doesn’t affect clinical care. Whether the meningococcal disease was serogroup A, B, C, Y or W-135 didn’t change how they cared for the sick individuals. Further testing was needed to show that all four cases had the exact same strain of serogroup B meningococcal disease.

That was a task for the Oregon State Public Health Laboratory; in an outbreak, it is the public health laboratory’s role to show cases are truly linked. As each case was determined to be meningococcal disease, the public health laboratory was contacted and serotyping began. While the public health lab’s confirmation that the patients were sick with group B meningococcal disease was enough information for CDC to green-light the vaccination effort, the Oregon State Public Health Laboratory dug even deeper. With Neisseria meningitidis cases such as the ones at the university, the Oregon state lab routinely uses pulsed-field gel electrophoresis (PFGE) to isolate the DNA fingerprint of each strain to show that everyone got the disease from the same source. That information could help epidemiologists identify the index case. “Using PFGE to fingerprint meningococcus is considered very risky, and it is very expensive, so many laboratories don’t do it,” explained Robert Vega, general microbiology manager at the Oregon state lab. “The risk associated with this is very real to us. Our staff is vaccinated against groups A, B, C, Y and W-135; we are well equipped and I have highly proficient staff.”

Once it was confirmed that the cases were group B meningococcal disease, CDC approved the Lane County Health Department and the University of Oregon to implement a massive effort to quickly vaccinate 22,000 students. The vaccination effort began on March 2 and within one week over 10,000 students had received the first dose of the vaccination. “We still have more students to reach, but we are working hard to make sure everyone is vaccinated,” said Dr. Luedtke. Quick treatment from clinical care providers and fast, accurate testing by the public health lab will hopefully mean that this is the beginning of the end of this outbreak.

Selection in a putative meningitis vaccine target

In Variation of the factor H-binding protein in Neisseria meningitidis, Carina Brehony in Martin Maiden's lab at Oxford investigated a group of outer membrane proteins in the bacterium responsible for meningococcal meningitis. To date, attempts to raise a vaccine against the common serogroup B meningococci have been frustrated by the low immunogenicity of the serogroup B capsular polysaccharide, despite success with serogroups A and C. Outer membrane proteins, such as factor H-binding protein (fHbp) may provide alternative targets for vaccine development.

However, fHbp is genetically diverse, and our investigation showed evidence of structuring into three groups. OmegaMap analyses of the three groups revealed a signature consistent with strong selection pressure for antigenic variability at the gene. Notably, there was clear evidence of diversifying selection at several previously discovered epitopes - positions in the protein targeted by antibodies during bacteria-killing immune response. (Analysis of one group is shown in the figure, with known epitopes marked).

While these observations are encouraging in terms of understanding the biology of pathogen antigens, a pressing question is how do we translate that understanding into practical vaccine design? Studies such as ours suggest a multi-component vaccine may be necessary to achieve broad coverage against serogroup B meningococci.