3 Syphilis and Vaccines

Let's learn a few things about Syphilis and the bacteria which causes it, Treponema pallidum. 

The reasons why I am looking at Syphilis on a tickborne-related illness blog are because a) it is spirochetal, b)  there is speculation on the similarities between Syphilis and Lyme Disease, c) some of the data on Syphilis may be informative in how one thinks about other infections or infection states, and last but not least d) some of the information given in this post provides background information for future posts.

So, what do we know about Syphilis? Nasty thing to get. I know I wouldn't want it. But then, I have Borrelia in me and that's bad enough; it's not even quite sure which Borrelia genotypes I'm hosting because tests I've had don't tell me.

Perhaps in the future, Dr. Ben Luft will develop a more accurate test, but I digress...

The overview

Syphilis is a sexually transmitted disease caused by the spirochetal bacterium Treponema pallidum subspecies pallidum. The primary route of transmission of syphilis is through sexual contact; however, it may also be transmitted from mother to fetus during pregnancy or at birth, resulting in congenital syphilis.

The signs and symptoms of syphilis vary depending on which of the four stages it presents in (primary, secondary, latent, and tertiary). The primary stage typically presents with a single chancre; secondary syphilis with a diffuse rash; latent with little to no symptoms; and tertiary with gummas, neurological, or cardiac symptoms. 

Blood tests are commonly used to diagnose syphilis; however those tests produce false negatives in 20-30 percent of primary syphilis cases, allowing for the possibility of ongoing transmission.[1]

Unfortunately, one of the reasons for inaccuracy in early detection of Syphilis is infectious disease specialist lack of access to darkfield microscopy. According to study author Deborah Dowell, MD, of the Centers for Disease Control and Prevention (CDC), "Eighty-one percent of our survey respondents did not have access to darkfield microscopy. These clinicians should treat presumptively if they suspect early syphilis in their patients." Dr. Dowell also notes that there is a clinical and public health need for a rapid point of care test to reliably diagnose primary syphilis.[1]

Right now, testing is a two-tier procedure:

Only if an EIA test is positive does the patient receive an RPR or VDRL test that is confirmatory, though it is a non-treponemal test that confirms the original reactivity. If the initial EIA test is negative, then the patient is considered to be uninfected with Syphilis. This is a serious problem due to the number of false negatives in the primary stage of infection.

The infection can be effectively treated with antibiotics in its earlier stages, specifically intramuscular penicillin G. There is also experimentation using oral azithromycin for treatment, though some strains are shown to be macrolide resistant.

Syphilis is believed to have infected 12 million people worldwide in 1999 with greater than 90% of cases in the developing world. Rates of infection have increased during the 2000s in many countries, including the United States.

The majority of cases have been concentrated in large cities, such as San Francisco, Baltimore, Miami, Washington, DC, and New York City, and scattered throughout the South. The highest numbers are found among inmates in correctional facilities, among men who have sex with men, and those co-infected with HIV.[2,3]

Syphilis infection increases the risk of transmitting and acquiring HIV infection. Not only are syphilis lesions a portal of entry for HIV but the immune cells that carry and are successful to the virus, macrophages and T lymphocytes, are found in abundance in syphilis lesions. There is also some experimental evidence for direct involvement of T. pallidum in facilitating HIV infection and progression.

Treponema pallidum

Treponema pallidum is a species of spirochaete bacterium with subspecies that cause treponemal diseases such as syphilis, bejel, pinta and yaws. It is not seen on a Gram stained smear because the organism is too thin.

What can we learn about it?

Electron micrograph of T. pallidum

Morphology 

• It is small, about 0.2 µm in diameter and between 6-15 µm in length. A human hair, in contrast is 40-50 µm.
  
• To get an idea of how small this really is, check out The Scale of the Universe and place the slider between the human body icon and the atom icon. Check out the red blood cell. Yeah, you're getting warm... that's how small it is.
  
• It requires dark field microscopy to see it. 
• It has regular, tight spirals and displays rotary, flexive and to-and-fro movements.
•  It has a cytoplasmic membrane enclosed by an outer membrane. 
• A thin layer of peptidoglycan is sandwiched between these membranes to give added stability.
•  The periplasmic space contains endoflagella that facilitate the characteristic motility.

Small genome

The genome of T. pallidum is much smaller (1.14 Mb) than that of many conventional Gramm-negative bacteria, for example, E. coli (4.6 Mb) and B. subtilis

Infectivity

• T. pallidum is transmitted by direct contact, usually sexual. 
• Infection is initiated when T. pallidum penetrates dermal microabrasions (small cuts) or intact mucous membranes (e.g. oral, ocular, nasal, vaginal). 
• Studies have shown that 16 to 30% of individuals who have had sexual contact with a syphilis-infected person become infected. Actual transmission rates can be higher. The 50% infectious dose is estimated to be only 57 organisms. 
• Upon initial infection the parasites prefer to multiply at the point of entry causing the inflammatory response and formation of characteristic chancre (an open sore).
• From the chancre the treponemes disseminate rapidly to the blood and lymphatics and make their way to different parts of the body including Central Nervous System (CNS). 
• T. pallidum has been shown to induce the production of matrix metalloproteinase-1 (MMP-1) in dermal cells. MMP-1 is involved in breaking down collagen, which may help T. pallidum to traverse the junctions between endothelial cells and penetrate tissues.
• The only known natural host of the bacterium is the human. Combined with transmission mode, this fact gives hope to a possibility of complete eradication of the syphilis in a future.

Challenges for research

• Treponema pallidum cannot be cultivated long term (more than 100-fold ~7 generations) in vitro. 
• In laboratory, T. pallidum can be only maintained by propagation in rabbits. 
• Because of fragility of its outer membrane, researchers are unable to modify the bacterium genetically in order to conduct experiments, which in many other bacteria clarified various aspects of their biology such as protein functions, mechanisms of virulence, and others.

The detailed nitty-gritty about Treponema pallidum

Metabolic deficiencies

• The bacterium lacks tricarboxic acid cycle enzymes and the electron transport chain. 
• T. pallidum depends upon glycolysis as the sole pathway for the synthesis ATP. 
• In addition, a pathway for amino acids and fatty acids synthesis as well as for metabolism of alternative carbon energy sources and for the synthesis of nucleotides and enzyme cofactors seems to be absent. 
• These traits suggest that the bacterium derives most essential macromolecules from its host (enzymes for interconversion of amino acids and fatty acids as well as homologs of transporters for a variety of amino acids are present). 
• Because the T. pallidum genome encodes no known homologs to porin proteins, it is unclear how nutrients are moved across the outer membrane into periplasmic space.

Slow multiplication rate
T. pallidum divides very slowly, doubling every 30-33 hours in vivo. In contrast, Nesseria gonorrhoeae divides approximately every 60 minutes, and E. coli every 20 min.

Limited stress response and heat tolerance

• The lack of enzymes that detoxify reactive oxigen species such as catalase and oxidase makes T. pallidum vulnerable to oxygen. 
• In a number of reports best survival occurred at low concentrations of oxygen (1-5%). 
• At least one of its enzymes is unstable at normal body temperature. 

Heat therapy for late neurosyphilis was introduced in 1918 by the Viennese psychiatrist Julius Wagner von Jauregg, a discovery for which he later won the Nobel Prize in Medicine. He inoculated patients with malaria pathogen and 10 to 12 febrile episodes later, treated them with quinine. 

The high temperatures induced by this regimen, along with other methods of raising body temperature, presumably killed T. pallidum in the CNS. Doctors reported high percentage of complete or partial remission of general paresis symptoms (although the treatment killed about 10% of patients - don't try IHT at home, kids!).

For a long time researchers tried to explain mechanisms underlying the natural course of the syphilis: recurring clinical manifestations separated by prolonged asymptomatic periods. In early 1970s it was believed that treponemes cause specific or generalized immunosuppression and are resistant to phagocytosis by macrophages and neutrophils. 

Later studies showed, however, that initial immune response of host to treponemal assault, though slow to develop, is rather robust, and as treponemes reach peak numbers, macrophages begin to infiltrate the lesions resulting in rapid clearance of overwhelming majority of the parasites from the tissue. However, some portion of the treponemes remains untouched and continues living in the host causing a persistent infection.

Lack of endo- and exotoxins

• The T. pallidum lacks liposaccharide (LPS), the endotoxin found in the outer membranes of many gram-negative bacteria. 
• The attachment of T. pallidum to cells does not harm the cells (no swelling or indentation). 
• The cultured cells survive for 5-7 days with actively motile attached treponemes in quantities up to 100 organisms per cell and remain viable. 
• This indicates that cytolytic enzymes or other cytotoxins most probably do not play a role in syphilis pathogenesis.

Invasion of "immune-privileged" tissues

T. pallidum penetrates a broad variety of tissues, including so-called "immune privileged": the central nervous system, eye, and placenta, where there is less surveillance by the host's innate immune system.

Ability to maintain infection with few organisms

T. pallidum may also exploit its slow metabolism to survive in tissues, even those that are not immune privileged. By maintaining infection with very few organisms in anatomical sites distant from one another, T. pallidum may prevent its clearance by failing to trigger the host's immune response, which was speculated to require a "critical antigenic mass".

Lack of surface antigens
Outer surfaces of bacterial pathogens are the first bacterial component to encounter the host and are often the targets of host adaptive immunity. One of most prominent features of T. pallidum is that its cell has only rare integral proteins in its outer membrane, approximately 1% of the number found in the outer membrane of E. coli. The rare T. pallidum outer membrane proteins are likely to be very important in interactions with the host; for this reason, their identity has been the subject of intense research.

Low iron requirements, ability to obtain sequestered iron
Iron sequestration is one of the important defense mechanisms used by the infected host. The host's transferrin and lactoferrin proteins bind free iron making it unavailable to bacteria and impairing their growth. T. pallidum may be able to acquire iron from these host proteins. It may also overcome the iron sequestration by using enzymes that need metals other than iron as their cofactors. In addition it lacks an electron transport chain, which is made up of enzymes that use iron as a cofactor, which decreases its overall demand for iron.

Resistance to macrophages in subpopulation of the pathogen
Opsonizing agents are serum components, antibodies, or the complement protein C3b, which make the pathogen recognizable to macrophages via specific cell surface receptors. T. pallidum antigens, including Tp92 andTprK, have been shown to induce production of opsonic antibodies. Antibodies against the VDRL (Venereal Disease Research Laboratory) antigen, a complex of cardiolipin, cholesterol, and lecithin, also increase the phagocytosis of T. pallidum by macrophages. Majority of treponemes that multiplied in quantities at the site of initial infection usually are cleared by macrophages. However, a small subpopulation of the organisms persists and appears to resist ingestion by macrophages. This phenomenon suggests that opsonic antibodies do not bind these organisms, thus allowing them to survive in the face of active immune clearance.

[To translate: Antibody opsonization is the process by which a pathogen is marked for ingestion and destruction by a phagocyte. Opsonization involves the binding of an opsonin antibody to a receptor on the pathogen's cell membrane. The immune system cannot always eliminate T. pallidum even though it has "recognized" the existence of the disease.]

Resistance to neutralization by antibodies
Besides opsonization, there are other functions of antibodies produced during T. pallidum infection. Antibodies developed against T. pallidum immobilize organisms and block them from binding the host's cells. Administration of whole serum and fractionated IgG from long-term-infected rabbits delays lesion formation in challenged rabbits, but lesions develop at the inoculation site within days of discontinuing the treatment. This demonstrates that specific antibody alone, while inhibitory to the establishment of lesions, is not sufficient to kill T. pallidum and prevent infection.

Orchestrated regulation of expression of antigens
Several genes that encode candidate outer membrane proteins belong to the tpr gene family which contains twelve genes that are divided into three subfamilies I, II, and III). The proteins encoded by tprF, tprI, and tprK are predicted to be located in the outer membrane.

Most of the proteins encoded by the tpr genes (the Tprs) elicit an immune response in experimental syphilis. Antibody responses arise at different times after infection: anti-TprK antibodies are seen as soon as 17 days postinfection and are robustly reactive at day 30, while antibodies against the members of subfamilies I and II often are not detectable until 45 days after infection and reach peak titers at day 60.

The time of development of antibodies to specific Tprs may reveal the timing of expression of the proteins that induced those antibodies. Regulation of expression of related proteins is referred to as phase variation and may be used by T. pallidum to down-regulate the expression of those Tprs against which an immune response has been mounted, while simultaneously up-regulating the expression of new Tprs, which are not recognized by the existing immune response. This strategy may help T. pallidum maintain chronic infection.

Antigenic variation of TprK protein
Recent studies identified TprK as a membrane-localized protein. The tprK gene and predicted protein amino acid sequences are characterized by seven discrete variable (V) regions that are separated by stretches of conserved sequences.

Diverse tprK sequences have been demonstrated between subpopulations of every T. pallidum strain within single host. DNA sequence cassettes that correspond to V-region sequences were discovered in an area of the T. pallidum chromosome separate from the tprK gene. These cassettes are potential sequence donors and are presumed to replace portions of V-region sequences in the tprK gene.

The TprK protein elicits both cellular and humoral immunity in infected animals. Antibodies to TprK that arise in response to T. pallidum infection are specifically targeted to the V regions. Very slight changes of the amino acid sequence in a V region can abrogate the ability of antibodies to bind the V region.

Thus, the host immunity may eliminate organisms that express TprK sequences against which specific antibodies have been developed. Generating new variation in TprK may help pathogens to escape immune recognition and sustain the chronic infection.

Vaccine Development

There is no vaccine for Syphilis. The outer membrane of T. pallidum has too few surface proteins for an antibody to be effective. Efforts to develop a safe and effective syphilis vaccine have been hindered by uncertainty about the relative importance of humoral and cellular mechanisms to protective immunity  and the fact that T. pallidum outer membrane proteins have not been unambiguously identified.

Some Abstracts On Vaccine Efforts

Assessment of cell-surface exposure and vaccinogenic potentials of Treponema pallidum candidate outer membrane proteins

Microbes and Infection

Volume 9, Issue 11, September 2007, Pages 1267-1275

T. pallidum is believed to be an extracellular pathogen and, as such, the identification of T. pallidum outer membrane proteins that could serve as targets for opsonic or bactericidal antibodies has remained a high research priority for vaccine development. However, the identification of T. pallidum outer membrane proteins has remained highly elusive. Recent studies and bioinformatics have implicated four treponemal proteins as potential outer membrane proteins (TP0155, TP0326, TP0483 and TP0956). Indirect immunofluorescence assays performed on treponemes encapsulated within agarose gel microdroplets failed to provide evidence that any of these four molecules were surface-exposed in T. pallidum. Second, recombinant fusion proteins corresponding to all four candidate outer membrane proteins were used separately, or in combination, to vaccinate New Zealand White rabbits. Despite achieving high titers (>1:50,000) of serum antibodies, none of the rabbits displayed chancre immunity after intradermal challenge with viable T. pallidum.

Progress towards an effective syphilis vaccine: the past, present and future

Authors: Cullen, Paul A; Cameron, Caroline E

Source: Expert Review of Vaccines, Volume 5, Number 1, February 2006 , pp. 67-80(14)

Syphilis is a disease caused by infection with the spirochetal pathogen Treponema pallidum subspp. pallidum. Despite intensive efforts, the unusual biology of T. pallidum has hindered progress towards the development of a vaccine to prevent infection. This review describes previous endeavors to develop a syphilis vaccine, outlines the key issues in the field and proposes new directions in the design of a T. pallidum vaccine. Following a brief overview of the disease symptoms, epidemiology, diagnosis and treatment, a case is put forward for the benefit of pursuing a syphilis vaccine. Relevant material concerning immunity to T. pallidum infection is summarized and evaluated, and pilot experiments describing the use of whole-cell bacterin vaccines and similar preparations are included. A detailed section concerning subunit vaccines is provided, incorporating discussions pertaining to relevant antigen selection, the identification of putative T. pallidum surface-exposed outer membrane proteins, factors hindering previous attempts to vaccinate with recombinant outer membrane proteins, problems and pitfalls of syphilis outer membrane protein-based vaccines, anti-attachment vaccines and the potential use of nonprotein subunit preparations as vaccinogens. Subsequently, critical aspects concerning vaccine antigen preparation and delivery are noted, including protein conformation, synergy, post-translational modifications, live attenuated organisms as vaccine vectors, prime–boost methodologies, adjuvant selection and immunization routes. Finally, animal models are discussed with particular reference to immunoprotection studies. A more thorough understanding of immunity to syphilis, a comprehensive assessment of the immunoprotective capacity of the putative surface-accessible antigens of T. pallidum and utilization of the latest advances in vaccine science should set the scene for future development of a syphilis vaccine.

LUKEHART SA; Interscience Conference on Antimicrobial Agents and Chemotherapy.

Abstr Intersci Conf Antimicrob Agents Chem other Intersci Conf Antimicrob Agents Chem other. 2000 Sep 17-20; 40: 540.

Univ. of Washington, Seattle, WA

Syphilis is a major public health problem in developing countries and in certain regions of the United States. It is estimated that there are twelve million new cases of syphilis per year, and at least 25 million infected persons worldwide. Syphilis is a recognized cause of perinatal morbidity and mortality and is a cofactor in acquisition and transmission of HIV infection. Despite the existence of safe and effective penicillin therapy, syphilis is unlikely to be controlled globally without an effective vaccine. Proof of concept for a syphilis vaccine was obtained in 1973, when Dr. James N. Miller demonstrated complete protection against infectious challenge in the rabbit model of experimental syphilis following immunization with gamma-irradiated Treponema pallidum. Since that time, numerous attempts to achieve protection using killed T. pallidum or isolated treponemal antigens have failed to yield satisfying results. Recent efforts have focussed on the identification of antigens that may be exposed on the surface of the intact treponeme, although definitive identification of such molecules has been difficult. This presentation will describe a number of candidate vaccine antigens that are currently under active investigation. Concerns relating to possible lack of heterologous cross-protection will be discussed in light of recent data regarding molecular heterogeneity of strains of T. pallidum. 

Biological Basis for Syphilis
Rebecca E. LaFond and Sheila A. Lukehart

Clinical Microbiology Reviews, January 2006, p. 29-49, Vol. 19, No. 1

Departments of Pathobiology, University of Washington, Seattle, Washington

Syphilis is a chronic sexually transmitted disease caused by Treponema pallidum subsp. pallidum. Clinical manifestations separate the disease into stages; late stages of disease are now uncommon compared to the preantibiotic era. T. pallidum has an unusually small genome and lacks genes that encode many metabolic functions and classical virulence factors. The organism is extremely sensitive to environmental conditions and has not been continuously cultivated in vitro. Nonetheless, T. pallidum is highly infectious and survives for decades in the untreated host. Early syphilis lesions result from the host's immune response to the treponemes. Bacterial clearance and resolution of early lesions results from a delayed hypersensitivity response, although some organisms escape to cause persistent infection. One factor contributing to T. pallidum's chronicity is the paucity of integral outer membrane proteins, rendering intact organisms virtually invisible to the immune system. Antigenic variation of TprK, a putative surface-exposed protein, is likely to contribute to immune evasion. T. pallidum remains exquisitely sensitive to penicillin, but macrolide resistance has recently been identified in a number of geographic regions. The development of a syphilis vaccine, thus far elusive, would have a significant positive impact on global health.

Ongoing Research of Dr. Caroline E. Cameron
2006-present University of Victoria, Victoria, BC

The laboratory of Dr. Caroline Cameron focuses upon spirochetal bacteria, with a specific focus on Treponema pallidum and Leptospira. The overall objective of the research is to identify and characterize molecules that are central to the pathogenesis of these important human pathogens.

The first main project investigates the extremely invasive nature of T. pallidum. Within our laboratory we have discovered several adhesins that contribute to T. pallidum attachment and a protease that is central to dissemination within the host. We are currently developing methods to target these proteins, with the ultimate goal of developing a prophylactic intervention to prevent the establishment of chronic infection.

The second major project within the laboratory involves the identification of novel diagnostic antigens, and the translation of this research into an improved syphilis diagnostic test.

We also perform proteomic analyses of Treponema pallidum to identify potential surface-exposed proteins within this unculturable pathogen.

The long-term objective of the research performed within the laboratory is to expand our knowledge of spirochete pathogenesis, which will in turn allow for the development of novel therapeutic reagents and/or preventative measures to combat infection. Future research will investigate the role of identified virulence factors in the pathogenesis of T. pallidum and Leptospira.

Citations

[1] Infectious Diseases Society of America (2009, October 23). Syphilis Survey Reveals Need For Accurate Testing For Early Infection. ScienceDaily. Retrieved January 13, 2011, from http://www.sciencedaily.com­ /releases/2009/10/091022122334.htm

[2] Stevenson, J., & Heath, M. (2006). Syphilis and HIV infections: An update. Dermatol Clin, 24(4), 497.

[3] Peterman, T., Heffelfinger, J., et al. (2005). The changing epidemiology of syphilis. Sex Transm Dis, 32(1), S4.

Other citations also expanded upon from: Metapathogen and http://en.wikipedia.org/wiki/Syphilis

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I'm guessing that for most of you, this is more than you ever knew about Syphilis or cared to know about it.

It's a start, really. There's more.

Think about everything you've read here, and anything you've read about Borrelia burgdorferi. There are differences and then there are similarities. They can't be treated interchangeably, but it is useful to look at the issues involved with both, where they do overlap, and how they've been approached in research. And also why each has been approached in research the way it has.