It’s hard to turn on the news at the moment and not hear about the latest emerging disease, Zika. The flavivirus* joins a cadre of infectious diseases spread by arthropod vectors – meaning the disease is passed between infected individuals via insect bites. A long list of microbes are spread this way, representing viral, bacterial, and protozoan pathogens: West Nile virus, Borrelia burgdorferi, Plasmodium species, Chikagunya virus, Trypanasoma species, Rickettsia species, yellow fever virus – the list goes on and on.
Some of the above microbial infections can be prevented with vaccines, but most cannot. The NIH has said patient trials for Zika virus vaccines may begin within 2016, but each vaccine requires multiple layers of testing before becoming widely used – not to mention the time it takes to find the best antigen, attenuated strain, or other immunization method. Dr. Erol Fikrig of Yale University studies vector-borne disease, which has given him insight into a unique aspect of microbial transmission often ignored in arboviruses and other vector-borne diseases.
When an insect such as a tick takes a blood meal, it doesn’t only draw out blood from its host, but it injects a bit of saliva. The saliva enters the bite site along with the microbe. What Fikrig wanted to know is whether “this saliva is merely a vehicle,” he says, “or does it have functional importance?” In other words, does tick spit affect the transmission of disease?
In this case, Fikrig was looking at transmission of the causative agent of Lyme disease, the spirochaete bacterium Borrelia burgdorferi. He first asked whether the presence of B. burgdorferi changed the gene expression of the tick salivary glands. Upregulation of specific genes might suggest the microbe was using tick salivary proteins to assist bacterial transition to a new host. Fikrig and his team of scientists found that indeed, ticks carrying the bacteria did have a different pattern of expression than those that had no bacteria. Further, his group found that several of these molecules in tick saliva bound to the bacteria.
The researchers then focused on one of these tick salivary proteins, Salp15, which was induced by Borrelia presence, to ask whether the protein interacts with the bacteria. By incubating purified Salp15 with the bacteria, they observed that the protein bound the outer bacterial surface, and they determined this interaction occurred through Salp15 interactions with the B. burgdorferi protein OspC. But why would B. burgdorferi coat itself in tick salivary proteins? This is where Fikrig used clever reasoning to test his idea.
“As the bacteria divide, the protein they are coated in doesn’t – there’s a finite amount of it,” Fikrig says “There’s a window in which this interaction occurs – how important is the Salp15-OspC interaction during this window?”
His group set about testing this in two key experiments. In the first, the group used RNAi to knock down expression of salp15 in the B. burgdorferi host tick, Ixodes scapularis. These ticks were fed a blood meal containing spirochaetes, and then later allowed to feed on an uninfected rodent host. The researchers saw that the ticks without Salp15 in their saliva didn’t transmit the bacteria to the new host as well as ticks that did have Sal15p.
Next, the researchers immunized mice with Salp15, after which they were exposed to bacteria-carrying ticks. The immunized mice had a much reduced infection compared to nonimmunized mice, supporting a role for Sal15p in establishing B. bergdorferi infection.
“All vaccines are pathogen based,” says Fikrig, a fact that has been true since Edward Jenner’s first inoculations using cowpox to prevent smallpox. But in this case, “the vector molecule can serve as a vaccine against the pathogen,” continues Fikrig.
Immunization with Salp15 alone may not be enough to stop B. burgdorferi transmission. However, this protein could be added to any microbial components to increase the efficacy of pathogen-based vaccines. To test this idea, Fikrig’s team took bacterial surface protein OspA antibodies, which can protect against infection in high doses, and administered them to mice at a diluted concentration too low to be protective. Some mice were then given antibodies against the tick protein Salp15, and all mice were then bitten by B. burgdorferi-carrying ticks. The researchers observed that the combination had a synergetic effect – mice given both types of antibodies were better protected than mice given only one. These two antigens, OspA and Salp15, might also work together to increase vaccine efficacy (see schematic, left).
Translating to other microbes and additional vectors
While this research is very promising for Lyme disease vaccine research, translating to other tick-borne diseases presents a challenge. “Salp15 immunization does not prevent feeding; the tick hasn't changed,” says Fikrig. Unless Salp15 plays a role in establishing infection for other microbes, immunizing against Sal15p is unlikely to ward off infection with Babesia microti or Anaplasma phagocytophilium, two other microbes carried by the Ixodes scapularis tick.
However, the phenomenon of tick immunity may hint at a way forward for pan-tick vaccine candidates. In 1938, George Trager of Rockefeller University observed that upon serial exposure to large number of ticks, a guinea pig experienced fewer and fewer bites over time. This so-called ‘tick immunity’ is in part due to the immune system learning to neutralize the numbing compounds released as the tick feeds. Immunity to such a compound may prevent tick bites, says Fikrig, but this is a double-edged sword: as the ticks are ‘rejected,’ their bites leave welts that, in the right circumstances, can develop ulcers and leave bite victims vulnerable to infection.
One reason ticks secrete numbing compounds into their hosts is in part because their blood meals last five days – much longer than a typical mosquito bite. “Ticks and mosquitos are very different animals because of their feed time differences,” among other differences, says Fikrig. Nevertheless, his research group is investigating molecules in mosquitos that may be involved in pathogen transmission. This work includes identification of the serine protease ClpA3 as a mosquito saliva component that enhances dissemination of Dengue virus into its host. ClpA3 is not a strong vaccine candidate due to similarity to human serine proteases (which, if used, could induce dangerous cross-reactivity), but this finding does optimistically demonstrate that mosquito saliva proteins participate in the infection process.
Fikrig’s group continues to work on Dengue virus, West Nile virus, and other flaviviruses (of which Zika virus is a member). He also hopes to extend this work to Plasmodium species, the causative agent of malaria. As the news has also emphasized recently, all mosquitos are not created equal: Aedes aegypti is not the same as Aedes albopictus is not the same as a number of Culex species. Each species has its own breeding habits, biting habits, preferred habitat, and geographic location. However, any compound able to combat any of these devastating diseases would be a huge progressive step toward life-saving preventative measures.
This work is being presented on Wednesday, February 10th, by Erol Fikrig during the 2016 ASM Biodefense and Emerging Diseases Research Meeting.
-- Julie Wolf
*Note: Zika was originally referred to as a filovirus, the taxonomic group of Ebola and Marburg virus. Zika is a flavivirus, categorized with dengue virus and West Nile virus.