**5.5 Credentialing polymorphisms**

It is clear that some antigens such as AMA1 and CSP have many polymorphic sites, while others such as EBA175 and MSP4 remain relatively conserved [8]. However, this is probably more a reflection of functional constraints or degree of immune exposure, than indicating their capacity to be an effective vaccine candidate. The challenge is how to determine which polymorphisms will be critical to vaccine design. Investigators have used different approaches to identify antigenically relevant polymorphisms, including three-dimensional structural modelling, immunological assays and mutational analysis [91, 97]. However, population genetic data - which may be easier to collect - can be highly informative.

When choosing which polymorphisms to consider for vaccine design it is important to define the allele frequencies for each polymorphic site. If the minor allele frequency (MAF)

Using Population Genetics to Guide Malaria Vaccine Design 251

**vii. Parasite population monitoring:** Even after an effective vaccine(s) has been developed and rolled out, sustained and intensive surveillance efforts will be essential to monitor population allele frequencies on a regular basis. This will identify significant fluctuations that might impact on vaccine efficacy or newly emerging alleles that may allow antigenic escape. With data obtained through such a surveillance system, formulations may then be adapted n a manner analogous to the Influenza vaccine, to

A broadly effective malaria vaccine continues to be one of the most sought after strategies in the fight against malaria, however the diversity of many of the parasite antigens currently being considered as subunit vaccine candidates is an obstacle that must be overcome. Natural malaria parasite populations are complex and dynamic communities, but modern molecular and analysis technologies are providing new ways to understand them. It is therefore important that researchers consider using population genetics to inform malaria vaccine development from target discovery through to vaccine design and to assess the

The authors are grateful for the support of the National Health and Medical Research Council (NHMRC) of Australia through project grants 488221 and 1003825. JB is supported by a Senior Research Fellowship; JCR by a Principal Research Fellowship; and FJIF by a Training Fellowship from the NHMRC. This work was made possible through Victorian State Government Operational Infrastructure Support and Australian Government NHMRC

[1] Moran, M., Guzman, J., Ropars, A, Jorgensen, M., McDonald, A., Potter, S. and Selassie,

[3] Doolan, D.L., C. Dobano, and J.K. Baird, *Acquired immunity to malaria.* Clin Microbiol

[4] Mu, J., et al., *Genome-wide variation and identification of vaccine targets in the Plasmodium* 

[5] Fluck, C., et al., *Strain-specific humoral response to a polymorphic malaria vaccine.* Infect

H. , *The Malaria Product Pipeline: Planning for the future*. 2007, The George Institute

evaluate any protective effects.

effects of clinical trials.

**7. Acknowledgments**

IRIISS.

**8. References** 

ensure continued protection against malaria.

for International Health: Sydney.

Immun, 2004. 72(11): p. 6300-5.

[2] Organization, W.H., *Malaria Vaccine Rainbow Table*. 2008.

Rev, 2009. 22(1): p. 13-36, Table of Contents.

*falciparum genome.* Nat Genet, 2007. 39(1): p. 126-30.

therefore it is not possible to assess whether any allele-specific protection has been induced in individuals receiving the vaccine. Given the relatively low cost (compared to conducting a vaccine trial) and ready availability of high throughput genotyping and NextGen sequencing technologies, this step should certainly be included in future vaccine trials and could even be done in retrospect if samples are still available. However, it is important to note that in some trials where the allele used to formulate the vaccine is found at low frequencies in the testing site, sample size may be too low to

is <10%, at least 90% of isolates will carry the predominant allele, and therefore a vaccine candidate carrying the major allele will target the majority of the parasite population. Mapping regions of significant balancing selection (where allele frequencies will be lowmedium) will identify regions that have accumulated polymorphisms as a result of being directly targeted by the immune response [236]. These analyses can help identify regions that are targets of functional immune responses. The analysis can be done by sliding window analyses of Tajima's D or measuring the HKAr for different protein domains. As balancing selection maintains balanced allele frequencies across different populations, it is also possible to use the interpopulation differentiation statistic, Wrights FST to measure differences in allele frequencies between populations. A low and insignificant Wrights FST value will demonstrate that allele frequencies are similar among populations and polymorphic regions potentially associated with immune escape [236, 242]. An additional important approach to determine key polymorphisms is by monitoring longitudinal infection dynamics in cohort studies, which may include vaccine trials [92]. If specific polymorphisms allow immune escape, then subsequent infections will carry alternative alleles to those of previous infections. Similarly, in vaccine trials, post-vaccination infections can be monitored for the presence of vaccine alleles. Antigenicity can then be determined by comparing allele frequencies in the vaccinated and control groups.

### **6. Conclusion: The way forward**

The malaria vaccine development pipeline has suffered many setbacks and complications over the decades since the first malaria vaccine was trialed. Nevertheless, it is our opinion that with carefully planned vaccine trials and an understanding of naturally circulating parasite diversity, made possible through technological advances in next generation sequencing and dedicated bioinformatics efforts, a broadly effective subunit malaria vaccine is achievable. With respect to developing such a vaccine we put forward the following recommendations for the design and testing of the next generation of malaria vaccines:


is <10%, at least 90% of isolates will carry the predominant allele, and therefore a vaccine candidate carrying the major allele will target the majority of the parasite population. Mapping regions of significant balancing selection (where allele frequencies will be lowmedium) will identify regions that have accumulated polymorphisms as a result of being directly targeted by the immune response [236]. These analyses can help identify regions that are targets of functional immune responses. The analysis can be done by sliding window analyses of Tajima's D or measuring the HKAr for different protein domains. As balancing selection maintains balanced allele frequencies across different populations, it is also possible to use the interpopulation differentiation statistic, Wrights FST to measure differences in allele frequencies between populations. A low and insignificant Wrights FST value will demonstrate that allele frequencies are similar among populations and polymorphic regions potentially associated with immune escape [236, 242]. An additional important approach to determine key polymorphisms is by monitoring longitudinal infection dynamics in cohort studies, which may include vaccine trials [92]. If specific polymorphisms allow immune escape, then subsequent infections will carry alternative alleles to those of previous infections. Similarly, in vaccine trials, post-vaccination infections can be monitored for the presence of vaccine alleles. Antigenicity can then be determined by

The malaria vaccine development pipeline has suffered many setbacks and complications over the decades since the first malaria vaccine was trialed. Nevertheless, it is our opinion that with carefully planned vaccine trials and an understanding of naturally circulating parasite diversity, made possible through technological advances in next generation sequencing and dedicated bioinformatics efforts, a broadly effective subunit malaria vaccine is achievable. With respect to developing such a vaccine we put forward the following recommendations for the design and testing of the next generation of malaria vaccines:

iv. **Prioritise vaccine candidates**: Many malaria vaccine candidate antigens have reached vaccine trials simply because they were discovered first. Genome sequencing and "omic" technologies have now identified several antigens that may have greater potential to induce protective immunity to a wide variety of parasite strains than these more traditional candidates. As the pipeline to clinical trials and eventual licensing is an arduous and vastly expensive journey, a thorough review of all candidates including those recently discovered and also in development needs to be done to identify those

v. **Prioritise vaccine alleles**: Dozens of population genetic studies have highlighted the issue of high levels of genetic diversity in candidate antigens and the low prevalence of the vaccine alleles currently being used, in natural parasite populations. Researchers now have the opportunity to mine already compiled datasets (e.g. [8]) or to rapidly sequence large numbers of field samples in vaccine testing sites to identify high frequency alleles for testing in single or multivalent vaccines. Notably, for a large number of leading malaria vaccine candidates under strong balancing selection, these

**vi. Allele-specific endpoints for vaccine trials:** Few malaria vaccine trials have evaluated allele frequencies either before, during, or after vaccine trials have taken place and

with the greatest potential to elicit protective immunity against malaria.

alleles may be common across populations.

comparing allele frequencies in the vaccinated and control groups.

**6. Conclusion: The way forward** 

therefore it is not possible to assess whether any allele-specific protection has been induced in individuals receiving the vaccine. Given the relatively low cost (compared to conducting a vaccine trial) and ready availability of high throughput genotyping and NextGen sequencing technologies, this step should certainly be included in future vaccine trials and could even be done in retrospect if samples are still available. However, it is important to note that in some trials where the allele used to formulate the vaccine is found at low frequencies in the testing site, sample size may be too low to evaluate any protective effects.

**vii. Parasite population monitoring:** Even after an effective vaccine(s) has been developed and rolled out, sustained and intensive surveillance efforts will be essential to monitor population allele frequencies on a regular basis. This will identify significant fluctuations that might impact on vaccine efficacy or newly emerging alleles that may allow antigenic escape. With data obtained through such a surveillance system, formulations may then be adapted n a manner analogous to the Influenza vaccine, to ensure continued protection against malaria.

A broadly effective malaria vaccine continues to be one of the most sought after strategies in the fight against malaria, however the diversity of many of the parasite antigens currently being considered as subunit vaccine candidates is an obstacle that must be overcome. Natural malaria parasite populations are complex and dynamic communities, but modern molecular and analysis technologies are providing new ways to understand them. It is therefore important that researchers consider using population genetics to inform malaria vaccine development from target discovery through to vaccine design and to assess the effects of clinical trials.
