Date Published: January 12, 2017
Publisher: Public Library of Science
Author(s): Thomas S. Churcher, Robert E. Sinden, Nick J. Edwards, Ian D. Poulton, Thomas W. Rampling, Patrick M. Brock, Jamie T. Griffin, Leanna M. Upton, Sara E. Zakutansky, Katarzyna A. Sala, Fiona Angrisano, Adrian V. S. Hill, Andrew M. Blagborough, Edward Wenger.
Over a century since Ronald Ross discovered that malaria is caused by the bite of an infectious mosquito it is still unclear how the number of parasites injected influences disease transmission. Currently it is assumed that all mosquitoes with salivary gland sporozoites are equally infectious irrespective of the number of parasites they harbour, though this has never been rigorously tested. Here we analyse >1000 experimental infections of humans and mice and demonstrate a dose-dependency for probability of infection and the length of the host pre-patent period. Mosquitoes with a higher numbers of sporozoites in their salivary glands following blood-feeding are more likely to have caused infection (and have done so quicker) than mosquitoes with fewer parasites. A similar dose response for the probability of infection was seen for humans given a pre-erythrocytic vaccine candidate targeting circumsporozoite protein (CSP), and in mice with and without transfusion of anti-CSP antibodies. These interventions prevented infection more efficiently from bites made by mosquitoes with fewer parasites. The importance of parasite number has widespread implications across malariology, ranging from our basic understanding of the parasite, how vaccines are evaluated and the way in which transmission should be measured in the field. It also provides direct evidence for why the only registered malaria vaccine RTS,S was partially effective in recent clinical trials.
Mosquito-to-human malaria transmission occurs when sporozoites from the salivary gland of the mosquito are injected into the skin during blood-feeding. Parasites then pass to the liver where they replicate, each sporozoite yielding many thousands of merozoites which go on to cause patent infection. Relatively little is known about the population dynamics of malaria between the bite of the infected mosquito and patency. It is currently assumed that the probability of mosquito-to-human transmission is determined simply by the presence of salivary gland sporozoites and not the total number of parasites. For example malaria transmission intensity and the human force of infection are measured using the entomological inoculation rate (EIR) the average number of infectious mosquito bites per person per year . EIR is calculated by multiplying the human biting rate by the proportion of mosquitoes with salivary gland sporozoites and therefore does not explicitly consider how heavily infected the mosquitoes are.
The association between the number of residual-sporozoites and the probability of infection was assessed in 47 vaccinated volunteers (challenged with Plasmodium falciparum using Anopheles stephensi mosquitoes), 9 of which became infected (Fig 1A). Though there was no difference in the mean (or total) residual-sporozoite number in mosquitoes biting persons who became infected or remained uninfected (S2 Table) a binomial model  shows that infection was significantly more likely from mosquitoes with >1000 residual-sporozoites (Fig 1B). The best fit model indicates that mosquitoes harbouring >1000 sporozoites had a per bite transmission probability of 9.2% (4.5%-16.0%) whilst those with a lower number of parasites did not measurably contribute to transmission (Fig 1B, see S3 Table for the results of the full model comparison). This model is used to estimate the probability that a volunteer became infected according to the number of residual-sporozoites in the bites they received (Fig 1C). Though there is still considerable variation, the predicted probability of infection is significantly higher in infected volunteers (p = 0.040) supporting the original analysis that mosquito parasite load influences the (per bite) probability of mosquito-to-human transmission. None of the 13 volunteers who received 0 or 1 bites from mosquitoes with >1000 residual-sporozoites became infected.
Together the human and rodent data provide strong evidence that mosquitoes with more salivary gland sporozoites post-feeding are more infectious. Though this cannot currently be tested directly in vivo, the most plausible explanation is that that mosquitoes with a higher number of residual-sporozoites injected more sporozoites into the skin during blood-feeding which increased the probability and the speed of the ensuing blood-stage infection. Whilst the probability of infection analysis could only be tested in humans who had been given a PEV, the consistent association between residual-sporozoites and the time to patency in naïve and vaccinated volunteers and the mouse experiments suggest that parasite intensity, and not just the number of infectious bites, is key to understanding the underlying biology of malaria transmission. The mouse experiments indicate a continuum of infection: with every sporozoite injected into the vertebrate increasing transmission.