Pathogens have perfected the art of manipulating our immune systems, and Salmonella enterica is one of the best. Prokarium is taking advantage of this natural expertise by re-engineering Salmonella into an efficient vaccine platform for a variety of infectious diseases. Earlier this month, the company secured funding with academic collaborator Robin Shattock (Imperial College London) from SynBICITE to push a new vaccine for Chlamydia through preclinical trials (see details at the end of the article).

In addition to being one of the most common sexually transmitted infections (more than 100 million new cases annually), Chlamydia is also the primary cause of female infertility, ectopic pregnancy and preventable blindness worldwide. It also causes pneumonia in infants. Although most cases are responsive to antibiotic treatment, containment of Chlamydia is difficult because so many carriers are asymptomatic. Up to 50% of infected men and 90% of infected women are unaware they are harboring the pathogen. There is currently no vaccine for Chlamydia but developing one is our best option for combatting this epidemic. Prokarium believe they can make this happen.

How Salmonella can vaccinate against Chlamydia

Wild type Salmonella enterica serovar Typhi infect the macrophages of our intestine, and then propagate within those cells. By secreting special effectors through syringe-like molecular machines, the bacteria prevent host cells from digesting them, allowing them thrive on the cell’s nutrients, causing tissue damage, evading immune response and making us feel terrible.Prokarium Ltd. has hacked this species into an efficient antigen delivery system. They acquired an attenuated S. enterica strain which had already been proven safe in multiple phase I and phase II trials. Prokarium engineered the strain to secrete antigens, from Chlamydia trachomatis in this case, instead of the effectors that allow it to persist within our macrophages.

The Vaxonella vaccine “will be taken orally and as it enters through the person’s gut lining it is naturally engulfed by the body’s own immune cells and only then triggered to produce the active vaccine, exactly where it is needed and without side effects in other parts of the body.” – Dr Ted Fjällman, CEO of Prokarium.The harmless bacterium is degraded after host infection, but not before directly secreting the antigen into the antigen presenting cells (APCs) of our gut. APCs display the antigen as a warning to the rest of the immune system, activating B and T cells that would recognize a Chlamydia infection. Immunological memory forms, and our body becomes protected from Chlamydia, without ever encountering the pathogen itself.Prokarium’s Vaxonella platform has broad applications. By incorporating different antigens, Prokarium is developing vaccines for not only Chlamydia, but also S. typhi, Clostridium, and enterotoxigenic E. coli (ETEC). Each protective strain is lyophilized, coated in a proprietary formulation, and taken orally.

Why Use Live Salmonella as a Vaccine?

Many of the vaccines we encounter are dead pathogens or their purified fragments. These antigens are mixed with an immune activating compound called an adjuvant and often injected under our skin. Why can’t we take this approach with Clostridium or Chlamydia?First, intramuscular and subcutaneous injection doesn’t effectively protect us from microscopic invaders in our intestine and other mucous membranes. Those regions are protected by a special compartment of our adaptive immune system called the mucosal immune system. To prime our mucosal immune system, a vaccination must be delivered directly to a mucus membrane. Salmonella, a pathogen that naturally targets immune cells in the mucus membrane, is an ideal delivery vehicle.Second, choosing an adjuvant to stimulate the immune system is often half the challenge of vaccine development. For mucosal vaccine development, adjuvant formulation can be a nightmare. Adjuvants must be immediately recognized as foreign and illicit a strong immune response. Injected vaccines have a more predictable adjuvant response because our bloodstream is a highly controlled environment. But our mucus membranes encounter a complex and unpredictable set of molecules, depending on our diet, environment and microbiome. To avoid constant inflammation, our bodies have a system of mucosal tolerance. With the wrong adjuvant, a mucus-targeted vaccine could promote mucosal tolerance to a pathogen, rather than protective immunity, making us more susceptible to infection. Live bacterial vectors like Salmonella have built in adjuvancy and circumvent this problem.

Photo: Salmonella bacteria, a common cause of food poisoning, invade an immune cell. Source: National Institutes of Health.

Prokarium is Making Salmonella a Safe Vaccine

One of the largest concerns with live oral vaccines is that the vaccine itself could be pathogenic in some patients. The S. enterica strain in the Vaxonella platform harbors genomic deletions that block its ability to synthesize aromatic compounds and construct one of the secretion systems it needs to thrive within human macrophages. Prokarium is also using clever techniques for avoiding antibiotic resistance in the strains they engineer. The gene encoding the secreted antigen is maintained on a DNA plasmid. Generally, plasmid loss is avoided using antibiotic treatment and an antibiotic resistance gene located on the plasmid. Prokarium’s plasmid contains an antibiotic resistance marker that is removed by a Salmonella recombinase after transformation. To maintain plasmid stability during vaccination without antibiotics, they employ their proprietary ORT-VAC system (Operator-Repressor Titration for Vaccines), which links plasmid presence with expression of an essential gene on the Salmonella genome. Avoiding antibiotic resistance provides another comforting safety measure for this live vaccine vector.

Funding and Future

SynbiCITE (Synthetic Biology Innovation - Commercial and Industrial Translation Engine) is contributing £377k to the £498k pre-clinical trials for the Vaxonella Chlamydia vaccine. SynbiCITE’s CEO, Dr Steve Chambers, announced that the new Prokarium vaccine was awarded funding because "the need for a Chlamydia vaccine is significant and the potential for this approach both for Chlamydia and as a model for treating other infections in the medium-term is very encouraging." Clinical trials are scheduled to begin as early as 2017. If successful, the technology will be an important step for treating the world’s most prevalent STI and Prokarium will have access to a $6 billion global market.