Bordetella pertussis Vaccine

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Before the invention of its vaccine, Bordetella pertussis was one of the most prevalent childhood illnesses. This prevalence, combined with its high mortality rate of 1 death per 10 cases, lead to more annual deaths than polio and measles together during the early 20th century [2]. The World Health Organization claims B. pertussis infection, commonly referred to as Whooping Cough, is responsible for the most preventable deaths due to illness worldwide [1]. The vaccine became widely available during the 1950s and 1960s, leading to a reduction in incidence of over 90% [1]. Recently the number of Bordetella pertussis cases has been on the rise, and several minor epidemics have broken out in highly vaccinated countries. This indicates that the vaccine may be failing [2]. Researchers have begun to investigate the sources of these failures.

Whooping Cough


Background


Whooping Cough is an infection of the respiratory tract most commonly caused by the bacterium Bordetella pertussis, a small gram-negative bacterium [1]. This bacterium non-invasively adheres to the mucosal lining of the tracheobronchial tree. B. parapertussis and B. bronchiseptica are two additional pathogens that can present with whooping cough symptoms; however, these infections tend to be far less severe in nature. The first accounts of the disease date back to the 16th century. Before the vaccine, cyclic epidemics occurred every 2-5 years [10]. The disease is most frequently diagnosed in children ages 1-5 and presents itself as a moderate to severe respiratory infection with characteristic “whooping” coughing fits followed by vomiting. The disease has an incubation period of 9-10 days and symptoms can persist several months after they first manifest [1]. When infants contract the disease, it can manifest as apnea and cyanosis without any cough whatsoever. Infants have the highest rate of mortality due to the pertussis infection, a rate of 160 deaths per 100,000 cases [2]. In older children and adults the disease often manifests in unexpected and varying ways, making it difficult to diagnose [7].


Pathogenesis
B. pertussis is a pathogen primarily transmitted via inhalation of airborne particles or through direct contact with infected discharge, primarily from the nose and throat. The course of the disease has three distinct stages: the catarrhal, paroxysmal, and convalescent stages [10]. The catarrhal stage is characterized by cold or flu-like symptoms and during this stage the disease is at its most contagious [1]. As B. pertussis bacteria infecting the lungs multiply, the symptoms worsen to include the convulsive, violent coughing fits the disease is named for. Other unpleasant symptoms such as narrowing of the glottis, vomiting, lymphocytosis, and overactive mucus production can also occur during this stage [10]. Finally the convalescent stage is the recovery stage. Recovery is often gradual, extending over as long as a month [1].

Figure portrays the crystal structure for the Bordetella pertussis toxin protein complex. PTX contains 5 distinct protein subunits and is the primary virulence factor of the B. pertussis pathogen. It was created by Takuma-sa from the work of Penelopy E Stein, Amechand Boohoo, Glen D Armstrong, Stephen A Cockle, Michel H Klein, and Randy J Read. [(http://commons.wikimedia.org/wiki/File:Pertussis_toxin_complex.png)], via Wikimedia Commons


Although the direct cause of progression from one stage to the next is not completely understood, many virulence factors have been identified, isolated, and studied. Pertussis toxin (PTX) is believed to be B. pertussis’ primary virulence factor and is responsible for many of its detrimental symptoms. The gene encoding PTX is found is several Bordetella strains including B. parapertussis, and B. bronchiseptica, in addition to B. pertussis; however, pertussis is the only bacteria capable of producing a functional form of the toxin. PTX has 5 subunits and is consistent with the A-B family of toxins. It is known to cause leukocytosis, splenomegaly, histamine sensitization, hypoglycemia, and hypoproteinemia. On the molecular level, it acts as a toxin that, when secreted, binds to receptors on the surface of host cells. It attacks cells by ribosylating G regulatory proteins to inhibit the target cell’s intracellular transduction pathway and blocking arachidonate release, calcium mobilization, and phosphatidylinositol hydrolysis, greatly debilitating the target cell [7]. Although pertussis toxin is the most prominant toxin, it is by far not the only damaging toxin secreted by B. pertussis. Dermonecrotoxin is a single-polypeptide, cytoplasmic, heat-labile toxin. When injected intradermally in mice, the toxin causes necrotic lesions and when injected intravenously, the toxin is lethal. In humans it is believed to cause inflammation, restriction of blood-flow, and lesions around pertussis colonies in the lungs. On the molecular level, it stimulates DNA and protein synthesis in host cells while suppressing advancement through the cell cycle, leading to polynucleation. Some other important, but less well-studied toxins include: Brk (Bordetella resistance to killing) proteins, which help pertussis defend against the host immune system, tracheal cytotoxin, which kills ciliated tracheal epithelial cells, and adenylate cyclase, which induces apoptosis in macrophages [10]. Finally, B. pertussis has a unique lipopolysaccharide (LPS) that differs from that of other Gram-negative bacteria in its lack of O antigen. It functions similarly to that of other Gram-negative bacteria in that it causes fever, is mitogenic and is toxic to host cells; however, the absence of the O antigen makes it more difficult for the hosts immune system to recognize and target effectively [10].

B. pertussis has a number of adhesins that anchor it to the epithelial lining of the host respiratory tract. Pertactin (PRN) is a 69-kDa membrane protein believed to play a role in attachment as it contains the RGD tripeptide, which is common in other bacterial adhesins; however, experimental support has yet to be found. It is, however, a potent immunogen, known to be a primary target for pertussis-specific T-cells. Unlike PTX, which is almost completely homogeneous across all strains, 13 variants of PRN have already been identified. Fimbriae are part of the operon cya. This operon has 5 genes, of which, only 3 are important in pertussis: fimB, fimC, and fimD. FimB is found in periplasm and FimC in the outer membrane. Their specific molecular roles are not fully understood; however, they are involved in anchorage to host cells and believed to be the vessel by which toxins are injected into host neutrophils and macrophages [10]. Filamentous hemagglutinin (FHA) is a 220-kDa outer membrane protein. It is one of the earliest proteins expressed, detectable within a few minutes of infection, and in known to have many functions. It is know to be an adhesin as FHA-deficient mutants cannot adhere to host cells in vivo or in vitro [10]. However, on the molecular level, it is known to bind to receptor type 3 (CR3) on the surface of host macrophages, which leads to a delayed T-cell response in mouse models. It also suppresses the Th1 cell-mediated response, which lowers host immunity [7]. Tracheal colonization factor and serum resistance factor also play important roles in B. pertussis' ability to adhere to, and form colonies within a host [10].

Current Vaccine Options


DTwP Vaccine
The DwP pertussis vaccine, or the whole-cell vaccine, was developed during the early 1900s using dead cells of the Bordetella pertussis bacteria. It became prevalent in many developed nations during the 1940s and 1950s and effectively brought the disease under control [8]. When it first came into use, the vaccine was delivered 3 times throughout early childhood. Because localized reactions tend to increase with age, as the immune system increases in strength, this vaccine is not approved for adolescents or adults. Currently, the vaccine is based on standardized strains of B. pertussis that are killed and often treated with formalin; however, the exact method of production varies among manufacturers. Each vaccine undergoes rigorous testing that assesses potency, toxicity, sterility, and bacterial concentration. Aluminum salts are always added to the wP vaccine as an adjuvant. Thiomersal and other preservatives may also be present in the vaccine. Commonly, the vaccine is paired with diphtheria and tetanus toxoids to form a combined vaccine. Unfortunately, this vaccine elicits a strong immune response that often leads to adverse reactions. The vast majority of these reactions are minor and temporary, such as redness and swelling at the injection site, slight fevers, and agitation, which occur in approximately 1 in 2 to 10 innoculations [1]. However, some individuals, when exposed to live or dead forms of this bacterium, have a severe reaction mediated through pro-inflammatory cytokine induction within the central nervous system. This response leads to extreme fevers and convulsive encephalopathic symptoms [6]. Although these effects are also temporary and occur in less than one in 100 injections [1], they have led to speculation on the safety of the vaccine [6].


DTaP Vaccine
Currently, the preferred vaccine in most industrialized nations is the acellular vaccine. Acellular vaccines are created using antigen proteins identified and isolated from a pathogen. In order for an acellular vaccine to be effective, it must provide an immune response similar to that triggered by direct contact with the pathogen itself. The earliest form of this vaccine was created in 1981 by a Japanese researcher. The current aP vaccine is made up of 5 different antigens: PT, FHA, PRN, and FIM type 2 and type 3 [1]. Animal models and human clinical trials were used to assess which virulence factors produce protective antigens upon exposure [10]. The vaccine differs significantly from one manufacturer to the next, as there are no standardized strains from which these antigens are purified from, and no standardization for the processes of purification, detoxification, or incorporation of adjuvants and preservatives. The components of the acellular vaccine are combined with diphtheria and tetanus toxoids. This vaccine is usually administered 5 times during childhood and once more during early adolescence. In order to combat the increased strength and consequently reaction of an older immune system, the “boosters” after the original inoculation contain lower concentrations of the antigens, in order to minimize their reactogenicity [1]. The aP vaccine has been successful because it effectively minimizes the adverse side effects so prevalent in the wP vaccine. When side effects do manifest they are less severe and much less frequent [6].

Vaccine Failure

Figure shows the rate of Diagnosed pertussis cases within the United States from the year 1990 to the year 2013 for 5 distinct age groups. It shows an increasing general trend for all age groups with a particularly dramatic increase in children, adolescents,and young adults. The rising rate of whooping cough in individuals not yet one year of age is worrisome as these individuals have the highest mortality rate associated with the disease. Used with the permission of Dr. Thomas Clark of the CDC.


Incidents
The first pertussis epidemic to strike the post-vaccination bliss occurred in the United States in the 1990s and early 2000s. It was peculiar in that it was found not in young children, its primary victim in the past, but in a disproportionately high number of adolescents. This number increased throughout the 1990s and by the year 2000, 7 out of 100,000 individuals, ages 11-19, was diagnosed with disease. This rate far exceeded that of children ages 1-6 or 7-10. Adolescents were also strongly affected during the epidemic spanning the years 2004 and 2005. Of the over 25,000 cases reported in each year, adolescents comprised 36% and 30% respectively. These two outbreaks led to the introduction of an additional pertussis booster injection during the early adolescent years of 11 or 12 [2]. The most recent epidemics occurred during 2010 and 2012 [3]. The 2010 outbreak was highly localized to California, in so much as 9,000 of the 27,550 cases reported nationwide were from this state. In this epidemic, the primary victims were children ages 7-10 years, the majority of whom had received all 5 recommended doses of the DTaP vaccine. The 2012 outbreak primarily affected Washington, with a statewide disease rate of 37.5 ill individuals in 100,000. The group that was affected most severely was children of age 10. Adolescents ages 13 and 14 also had disproportionately high rates of incidence. The nationwide caseload of pertussis cases totaled 48,277, the most cases seen in America since the year 1959 [2].

The United States has not been the only industrialized nation affected in recent years. England and Wales also experienced a major outbreak in 2012. This epidemic claimed the lives of 14 infants [4]. Increased incidence rates among adolescents and adults leads to a greater chance of transmittance to unprotected infants who will ultimately face the greatest risk from the bacteria. Researchers believe that adults and adolescents have become the reservoir for the disease, from which infants with immature immune systems are at risk [10].


Differences in Vaccine Efficacy
As of 2010, the World Health Organization reported a pooled efficacy of 78%, with a range of 46% to 92%, for the DTwP vaccine. They reported a similar efficacy for the DTaP vaccine, in all of its 5-component, 4-component, and 3-component versions [1]. However, with the many recent outbreaks, scientists have returned to the Bordetella pertussis vaccines to assess their long-term efficacy.

Results of the 2010-2011 study on adolescents born between 1994 and 1999. Shows a lower incidence of pertussis infection among individuals who received DTwP for their first 4 childhood vaccinations when compared to individuals who had received all DTaP, or a mixture of DTwp and DTaP, suggesting DTwP provides stronger and longer-lasting immunity. This figure was created using the data from Comparative Effectiveness of Acellular Versus Whole-Cell Pertussis Vaccines in Teenagers. http://pediatrics.aappublications.org/content/131/6/e1716

Some studies chose to look at the efficacy of the vaccines directly. One study, performed at the Northern California Kaiser Permanente Vaccine Study Center, in 2010 and 2011, looked at the vaccination records of fully vaccinated adolescents born between the years 1994 and 1999. These were the years during which the transition from the DTwP to DTaP vaccine was made in the United States. Researchers compared relative incidence rates among the groups who had received varying proportions of DTaP to DTwP vaccines. Groups that received more acellular vaccines had a much higher rate of testing positive for the pertussis bacterium [3], suggesting the DTaP vaccine does not instill comparable immunity to the DTwP vaccine. Another recent human trial looked at the transmission of pertussis to children who themselves had already received a full set of three DTwP vaccinations. The efficacy of the DTwP vaccine against transmission was found to be 85%, whereas that of the DTaP vaccine was only 6% [10]. Although it is recognized DTaP protects against contraction of the disease and leads to less severe symptoms upon infection, these results suggest it does not prevent the spreading of the disease.

Other studies chose to focus on how each vaccine affects the human immune system. Through this research it was discovered that B. pertussis is able to survive within host macrophages and neutrophils [10]. As such, humoral immunity, or antibody production, may not be sufficient for long-term immunity. Conversely, cell-mediated immunity, or the mobilization of phagocytes and cytotoxic T-lymphocytes, appears to be the central unit in long-term pertussis immunity. Natural infection and convalescence from whooping cough produces a predominantly cell-mediated response. The wP vaccine was found to elicit a similar response; however, the aP vaccine elicited a very strong humoral response instead [10]. The differences in how the immune system primarily responds to the two vaccines could account for the variability in immunity they bestow.


Other Hypotheses for the Failures
There are several alternate hypotheses for the recent pertussis outbreaks. The strains used in laboratories across the nation were isolated over 70 years ago when the first vaccines were being researched. Bacteria are some of the fastest evolving species on Earth due to the speed with which they replicate their DNA and divide. It is hypothesized that wild pertussis and laboratory strains may have diverged significantly since this isolation event, as bacteria kept in a laboratory setting experience different selective pressures than those in their natural habitat. The recent epidemics have also created a necessity for improved diagnosis in adolescents and adults, where previously, many cases were left undiagnosed, or were improperly diagnosed. Increased diagnosis could be a confounding factor contributing to elevated case numbers [10].

Vaccines offer protection to the community, as well as to the individual, through herd immunity. Herd immunity occurs when the vast majority of individuals in a population are vaccinated against a disease. It offers indirect immunity to individuals who are not vaccinated by limiting the disease’s prevalence within the population. In the case of whooping cough, herd immunity protects infants who are most susceptible to the illness, but are too young to be inoculated. The pertussis vaccine, be it the DTwP or DTaP vaccine, has never offered perfect immunity. The level of immunity and duration of immunity has always been variable, and neither vaccine is believed to offer immunity comparable to that naturally acquired from contraction and convalescence of the disease itself. As such, some researchers believe the current outbreaks may be due to waning herd immunity. In other words, the first generations who grew up with the DTwP vaccine and avoided childhood infection by pertussis may have had no better immunity than the current generations growing up with DTaP; however, because a vast majority of the population had strong immunity gained through contraction of the illness, these individuals were protected through herd immunity. Unfortunately, that is no longer the case, as currently, the vast majority of individuals within industrialized nations have the weakened immunity provided by vaccines so there is no herd immunity remaining and an overall increase in incidence of infection. A team of researchers in England evaluated the epidemics using computer models to simulate disease propagation through a population as individual immunity changed over time and found projected incidence of infection that are consistent with current rates. Their results indicated that the current outbreaks are a natural response to herd immunity weakened by an imperfect vaccine [4].

The Search for Improvement


Although a loss of immunity is a terrifying concept, the current B. pertussis vaccine is still preferable to no vaccine at all. The primary targets for pertussis infection are small children, who also have the most dramatic cases and detrimental symptoms [1]. The DTaP vaccine is successful at protecting these young children from the disease. The primary concern with the DTaP vaccine is that an undiagnosed ill mother, or other family member, could pass the disease to her newborn infant, too young to receive vaccinations. Several measures have been proposed to ensure these situations do not occur [10]. For one, an increased awareness has led to an increase in diagnosis in older individuals [1]. Additionally studies are currently being carried out to evaluate the potential benefits of administering a DTaP booster shot to pregnant mothers. This injection would reactivate the mother's immune system to produce antibodies targeting the B. pertussis pathogen. It is hypothesized that these antibodies would pass through the placenta and provide the newborn baby with increased passive natal protection in that important first year of life [10].

Plenty research is also underway to improve the B. pertussis vaccine. An ideal vaccine would neutralize or abrogate the effects of every virulence factor and would also induce cell-mediated immunity, which is important for imparting long-term immunity [10]. Many hypotheses exist as to how this would best be accomplished. Some scientists advocate for a streamlining of the current DTaP vaccine to focus on the virulence factors known to induce the most aggressive immune response [5]. Others believe a more stringent booster regiment is necessary to reinstate herd immunity [4]. Still others dedicate their time to exploring the less well understood virulence factors to evaluate their potential [5]. Through further research and increased awareness, B. pertussis could lose its title as the number one cause of preventable death due to infectious disease worldwide.

References

1. "Pertussis Vaccines: WHO Position Paper." Weekly Epidemiological Record 85.40 (2010): 385-400. Academic Search Premier. Web. 26 Feb. 2015.

2. Clark, Thomas A. "Changing Pertussis Epidemiology: Everything Old is New Again." The Journal of Infectious Diseases 209.7 (2014): 978-981. Web. 26 Feb. 2015.

3. Klein, Nicola P., Joan Bartlett, Bruce Fireman, Ali Rowhani-Rahbar, and Roger Baxter. "Comparative Effectiveness of Acellular Versus Whole-Cell Pertussis Vaccines in Teenagers." Pediatrics. 131.6 (2013): 1716-1722. Web. 26 Feb. 2015.

4. Riolo, Maria A., Aaron A. King, and Pejman Rohani. "Can vaccine legacy explain the British pertussis resurgence?" Vaccine. 31(2013): 5903-5908. Web. 26 Feb. 2015.

5. Robbins, John B., Rachel Schneerson, Joanna Kubler-Kielb, Jerry M. Keith, Birger Trollfors, Evgeny Vinogradov, and Joseph Shiloach. "Toward a new vaccine for pertussis." PNAS. 111.9 (2014): 3213-3216. Web. 26 Feb. 2015.

6. Donnelly, Sheila, Christine E. Loscher, Marina A. Lynch, and Kingston H.G. Mills. “Whole-Cell but Not Acellular Pertussis Vaccines Induce Convulsive Activity in Mice: Evidence of a Role for Toxin-Induced Interleukin-1β in a New Murine Model for Analysis of Neuronal Side Effects of Vaccination.” Infection and Immunity. 69.7 (2001): 4217-4223. Web. 15 Mar. 2015.

7. Mattoo, Seema and James D. Cherry. “Molecular Pathogenesis, Epidemiology, and Clinical Manifestations of Respiratory Infections Due to Bordetella pertussis and other Bordetella Subspecies.” Clinical Microbiology Reviews. 18.2 (2005): 326-382. Web. 15 Mar. 2015.

8. Fine, Paul E. M. and Jacqueline A. Clark. “Reflections on the Efficacy of Pertussis Vaccines.” Reviews of Infectios Diseases. 9.5 (1987): 866-883. Web. 15 Mar. 2015.

9. Gustafsson, Lennart, Hans O. Hallander, Patrick Olin, Elisabet Reizenstein, and Jann Storsaeter. “A Controlled Trial of a Two-Component Acellular, a Five-Component Acellular, and a Whole-Cell Pertussis Vaccine.” New England Journal of Medicine. 334 (1996): 349-356. Web. 16 Mar. 2015.

10. Marzouqi, Ibrahim, Peter Richmond, Scott Fry, John Wetherall, and Trilochan Mukkur. “Development of improved vaccines against whooping cough: Curent status.” Human Vaccines. 6.7 (2010): 543-553. Web. 16 Mar. 2015.

Edited by Kristina Millar, a student of Nora Sullivan in BIOL168L (Microbiology) in The Keck Science Department of the Claremont Colleges Spring 2015.