Keywords

1 Current Pertussis Vaccines

Pertussis, also known as whooping cough, is a highly contagious respiratory disease mainly caused by Bordetella pertussis, a Gram-negative bacterium. This disease that causes uncontrollable violent coughing, affects all ages, being the most vulnerable the infants under 6 months of age (Stefanelli et al. 2017). The best way to prevent pertussis is to get vaccinated. The first experimentations with vaccines began after Jules Bordet and Octave Gengou of the Pasteur Institute of Brussels identified the etiological agent in 1906; these vaccines were made from killed whole-cell B. pertussis. In ensuing years, such type of vaccine (whole-cell vaccine, wP) was used in children in different countries. Thorvald Madsen was the first to describe the use of a wP vaccine on a large scale (Madsen 1933). Madsen’s vaccine successfully controlled two outbreaks in the Faroe Islands, however some deaths within 48 h of immunization were reported (Madsen 1933). Noteworthy at that time physicians used the vaccine as either a therapeutic or a prophylactic formulation and in both cases the vaccine was given in three injections intramuscularly or subcutaneously with intervals of three to 4 days (Madsen 1933). Madsen T. in his work summarized some reports that concluded sic…if the vaccine is given early in the catarrhal stage the vaccine will have a good effect; the later the vaccine is given in the convulsive stage, the less effect can be expected. This appears from the reports of most of the Danish officers of Health and also is the consensus of the Danish pediatric society (Madsen 1933). Louis Sauer of Northwestern University Medical School, Chicago, described minor reactions to a whole-cell pertussis vaccine being used in the United States as an adjuvanted combined vaccine (Sauer 1948). Pearl Kendrick of the State of Michigan Health Department further refined wP vaccines. She and Grace Eldering combined this improved killed vaccine with diphtheria and tetanus toxoids to produce the diphtheria-tetanus-pertussis (DTP) and used it in children (Kendrick 1936). The Committee on Infectious Diseases of the American Academy of Pediatrics suggested in 1944 and recommended in 1947 the routine use of pertussis vaccine in the form of the DTP combination. The use of this vaccine was then expanded to other countries. The coverages of pertussis vaccine were improved when the Expanded Program on Immunization (EPI) was established in 1974. The mission of the EPI is to develop and expand immunization programs throughout the world. In particular, in 1977, the goal was set to make immunization against diphtheria, pertussis, tetanus, poliomyelitis, measles and tuberculosis available to every child in the world by 1990. The massive pertussis vaccination dramatically reduced the morbidity and mortality associated with the disease (Table 1). After this important achievement in the control of the disease, unfortunately, doubts about the safety of wP vaccines began to arise and this led to a decrease in the acceptance of this type of formulation by the population and even in some countries its use was rejected (Klein 2014; Romanus et al. 1987). The first published reports on irreversible brain damage after whole-cell pertussis vaccination was described by Brody and Sorley. These reports led to the first warnings that pertussis vaccine should not be administered to those with a known neurologic disorder (Brody and Sorley 1947). In Great Britain, concerns on the safety of this vaccine were widely publicized in the popular press and because of that the proportion of children vaccinated against pertussis diminished (Kulenkampff et al. 1974). The adverse reactions ranged from local reactions (redness, swelling, and pain at the injection site) to systemic reactions (fever, persistent crying and, in rare cases encephalopathy) were reported in other countries (Klein 2014; Romanus et al. 1987). Concerns about safety finally led to the development of component (acellular) pertussis vaccines that are associated with a lower frequency of adverse reactions (Sato and Sato 1985; Edwards and Karzon 1990). These second-generation of pertussis vaccines, referred to as aP vaccines, are constituted of purified B. pertussis antigens combined with diphtheria and tetanus toxoids. The first acellular vaccine that was developed in Japan in 1970 consisted of two proteins: pertussis toxin (PTx) and filamentous haemagglutinin (FHA) (Sato and Sato 1985). Field trials showed that component vaccine was as effective as and produced less side-effects than did conventional whole-cell vaccine (Sato et al. 1984). The vaccine has been used for mass immunization in Japan since 1981 and was highly effective in preventing pertussis disease. In 1994 the efficacy for two, three-component acellular, pertussis vaccines containing inactivated PTx, FHA, and pertactin (PRN), and one five-component acellular pertussis vaccine containing the same components plus fimbriae 2 and 3 was compared with a UK whole-cell vaccine (Olin et al. 1997). This study demonstrated that the wP vaccine and the five-component aP vaccine had similar efficacy against culture-confirmed typical pertussis, defined by at least 21 days of paroxysmal cough. The authors also found that the three-component acellular vaccine was less effective than the five-component-vaccine and the whole-cell vaccines against culture-confirmed pertussis when all cases irrespective of the duration of severity of cough, were included in the analysis (Olin et al. 1997). Thus, though there was no compelling evidence to support that wP vaccines should not be used, the aP vaccines began to be broadly accepted because of their lower reactogenicity, especially in industrialized countries where wP vaccines of the primary series (3 doses in infancy) was replaced by aP vaccine (Table 1). Currently, US and most of the EU countries use only aP vaccines (Table 1). The aP formulations restored people’s confidence in pertussis-containing vaccines, and the infection was controlled for several years. Notwithstanding, during the last decades the epidemiology of pertussis has changed (Clark 2014; Tan et al. 2015) with several major outbreaks occurring, the incidence of which not only indicated a waning immunity but also demonstrated that the wP vaccines gave children a longer lasting immunity than aP (Klein et al. 2013; Witt et al. 2012; Sheridan et al. 2012). Furthermore, the risk of pertussis was increased in schoolchildren and adolescents vaccinated exclusively with aP compared to those receiving at least one wP dose (Witt et al. 2013; Sheridan et al. 2012). This difference could result from the weaker immune response induced by aP vaccines (Mills et al. 2014): while aP vaccines mainly induce a Th2-skewed response (Ryan et al. 1998), wP vaccines induce a robust Th1 profile and the proliferation of respiratory tissue-resident memory CD4 T cells (Brummelman et al. 2015; Wilk and Mills 2018). Therefore, the aP vaccine induced immunity shows a more rapid decay and possibly a reduced impact on transmission compared with currently available wP vaccines (Tartof et al. 2013; McGirr et al. 2013). In addition to the waning of immunity induced by vaccination, in particular with aP vaccines (Koepke et al. 2014; McGirr and Fisman 2015), pathogen adaptation to escape vaccine induced immunity (King et al. 2001; Mooi et al. 2001; Mäkelä 2000; David et al. 2004; He et al. 2003; Bottero et al. 2007; Gzyl et al. 2004; Bowden et al. 2016), and the failure of pertussis vaccines, in particular aP vaccines, to prevent infection and spread of B. pertussis were also proposed to explain the resurgence of the disease. Regarding pathogen evolution, the first reports were related to polymorphism in genes coding for proteins included in the vaccine (PRN and PTx among others) (Mooi et al. 1998) and later in the pertussis toxin promoter (ptxP) (Advani et al. 2011; Kallonen et al. 2012). Recently, there has been an increase in B. pertussis isolates that do not produce some of the vaccine antigens (Lam et al. 2014; Barkoff et al. 2019). It has been proposed that the loss of this vaccine antigen probably provides a selective advantage for bacterial survival in populations vaccinated with aP vaccines (Martin et al. 2015). Commercial aP vaccines containing PTx, PRN and FHA are not as effective as expected in controlling the infection caused by the recent circulating bacteria that do not express PRN (Hegerle et al. 2014). Moreover, recently it was demonstrated in a mixed infection mouse model that PRN deficient B. pertussis strain colonizes the respiratory tract of aP immunized mice more effectively than the PRN positive strain (Safarchi et al. 2015).

Table 1 Number of reported cases of pertussis and type of pertussis vaccine used in different regions of the world (data extracted from WHO public information)
Full size table

Under this context, in 2015 the Strategic Advisory Group of Experts on immunization expressed concerns regarding the resurgence of pertussis in certain industrialized countries despite high aP-vaccine coverage (Meeting of the Strategic Advisory Group of Experts on immunization 2015). The switch from wP to aP for primary infant immunization was proposed as, at least partially responsible for that resurgence (Table 1, see reported cases of European Region). The World Health Organization (WHO) therefore recommended that the switch be considered only if, in the national immunization schedules, large numbers of doses including several boosters can be assured. Countries currently using aP vaccines may continue using them, but should consider the need for additional booster doses and strategies to prevent early-childhood mortality upon pertussis resurgence. In fact, the WHO published a position paper on this subject and wrote the following:

A switch from wP to aP vaccines for primary infant immunization should only be considered if the inclusion in the national immunization schedules of additional periodic booster or maternal immunization can be assured and sustained (Pertussis vaccines: WHO position paper, August 2015—Recommendations 2016).

National programmes currently using aP vaccine may continue using this vaccine but should consider the need for additional booster doses and strategies to prevent early childhood mortality such as maternal immunization in case of resurgence of pertussis (Pertussis vaccines: WHO position paper, August 2015—Recommendations 2016).

2 New Pertussis Vaccines

Pertussis vaccines are currently on the agenda due to the worrying increase of pertussis cases detected in different countries. There are an estimated 24.1 million cases of the disease and approximately 160,700 deaths occurring worldwide every year in children younger than 5 years of age (Yeung et al. 2017). It is very clear that the non-use of the current pertussis vaccines would lead to an even more challenging epidemiological scenario and for this reason the current vaccine administration and surveillance of the disease should be improved while new vaccines are being developed. The development of a new pertussis vaccine is a difficult task to achieve since no absolute correlate for protection exists, however there are enough data from animal models and human studies showing that although antibodies may mediate protection, Th1 and Th17 cellular responses and tissue resident memory (TRM) response are responsible for long-lasting protection (Mills et al. 2014). To induce or drive a Th1, Th17 and TRM response, different approaches have already been proposed (Allen and Mills 2014; Mielcarek et al. 2006; Dias et al. 2013). In the next section, the main approaches used so far for the development of new vaccines are discussed.

3 Live Attenuated Vaccine

The most advanced novel pertussis vaccine candidate is that developed by Locht et al. in Lille, France (Thorstensson et al. 2014; Mielcarek et al. 2010; Feunou et al. 2010; Skerry et al. 2009). This vaccine candidate, referred as BPZE1, and consisting in a live attenuated bacterial strain, (Locht 2014) was shown to be immunogenic and protective in mice and baboons after intranasal administration (Locht 2016, 2017). In mice a single nasal administration of BPZE1, but not a high dose of current commercial aP vaccine, induced B. pertussis-specific secretory IgA in the nasal cavity, and transfer of the nasal IgA was able to protect recipient mice against nasal colonization after B. pertussis challenge (Solans and Locht 2018). Though no protection experiments have yet been performed with BPZE1 against circulating bacteria, other interesting findings have already reported. It was detected that BPZE1 vaccine was able to induce CD4+CD69+CD103+ TRM cells in the nasal mucosa of mice, and these cells produced high levels of IL-17 and appreciable levels of IFN-γ. Thus, BPZE1 protects mice against nasal infection by virulent B. pertussis via an IL-17-dependent and sIgA-mediated mechanism (Solans and Locht 2018; Fedele et al. 2011). Moreover, recently a double-blind, placebo-controlled, dose-escalating study of BPZE1 given intranasally for the first time to human volunteers was performed as the first trial of a live attenuated bacterial vaccine against pertussis. In this study, 12 subjects per dose group received different quantities of colony-forming units as droplets with half of the dose in each nostril and 12 subjects received the diluent (control group) (Thorstensson et al. 2014). Local and systemic safety and immune responses were assessed during 6 months, and nasopharyngeal colonization with BPZE1 was determined with repeated cultures during the first 4 weeks after vaccination. In this trial, the vaccine candidate was found safe in young human adults, able to transiently colonize the human nasopharynx, and to induce antibodies to PTx, FHA, PRN and fimbriae after a single nasal administration (Thorstensson et al. 2014). This vaccine candidate is currently entering a clinical phase II trial.

4 Less Reactogenic Whole Cell Vaccine

The major cause of wP vaccine reactions is associated to the endotoxin which is a lipo-oligosaccharide (LOS) and because of that attempts were made to detoxify wP vaccines. Researchers at the Institute Butantan in São Paulo, Brazil, diminished the endotoxicity of the wP vaccine by performing a chemical extraction of LOS from the outer membrane (Dias et al. 2013). Chemical extraction of LOS resulted in a significant decrease in endotoxin content without affecting the integrity of the product. This development, however, raises doubts because with the LOS extraction the adjuvant capacity associated with this molecule would also be decreasing. Other alternative strategies to LOS removal are being sought, specifically a consortium of researchers proposed to work on structural changes of the molecule (on the LipidA) in order to retain de beneficial effects induced by the molecule but eliminating its reactogenicity. The results on this strategy have not yet been disclosed.

5 Acellular Pertussis Vaccines Containing Recombinant Inactivated Pertussis Toxin

The safety and superior immunogenicity of 9 K/129G genetically detoxified PTx (rPT) was demonstrated long time ago (Rappuoli 1999; Podda et al. 1993). Under this context, BioNet-Asia developed a new rPT-expressing B. pertussis strain (Buasri et al. 2012). This strain generated increased amounts of rPT compared to wild type strain and strains used in vaccine production and the purified rPT did not show any toxicity (Buasri et al. 2012). Thus, Bionet formulated a new acellular vaccine containing the recombinant genetically detoxified Pertussis Toxin (PTgen), FHA and PRN and presented the results of the first clinical study of this recombinant aP vaccine formulated alone or in combination with tetanus and diphtheria toxoids. For the phase I/II trial, 60 subjects (20 per each vaccine group) were enrolled and included in the safety analysis. This first-in-human study showed that BioNet’s PTgen-containing vaccine has a similar reactogenicity and safety profile than the Adacel® acellular vaccine. Moreover, the high immunogenicity of PTgen in adults was demonstrated Sirivichayakul et al. (2016). The results were consistent with previous studies that demonstrated high and sustained efficacy of rPT-containing aP vaccines in infants (Seubert et al. 2014). Recent findings on the ability of rPT-containing acellular vaccine to induce memory response make a significant difference with current acellular vaccines that include chemically detoxified components in terms of long-term protection. Specifically, the authors reported that the boosting of aP-primed adolescents with recombinant-aP induced higher anti-PTx and PTx-neutralizing responses than the current aP vaccine and increased PTx-specific memory B cells (Blanchard Rohner et al. 2018). These new acellular vaccines can thus overcome one of the weaknesses of current acellular vaccines: the rapid loss of induced immunity. However, it remains to study the protection capacity of this vaccine against current circulating bacteria and the selection pressure that this type of vaccine would exert on the circulating bacterial population. This last aspect, in principle, would not be solved with the recombinant acellular vaccine, since it is constituted by the same few immunogens as the current acellular vaccines.

6 New Antigens and Adjuvants for aP Formulations

The incorporation of novel antigens derived from B. pertussis to improve the current aP vaccines has also been explored. The B. pertussis adenylate cyclase toxin (Cheung et al. 2006), the serum-resistance autotransporter protein BrkA (Marr et al. 2008) and the iron-regulated B. pertussis proteins (Alvarez Hayes et al. 2013) among others, have been proposed as a protective antigen. Though none of these antigens alone offered significant protection against B. pertussis infection in an intranasal challenge model, when combined with acellular pertussis vaccine, they conferred improved protection over the acellular vaccine alone. The combination of all these immunogens together with the current acellular vaccines could be an attractive proposal to reduce the selection pressure of the current acellular vaccines by offering a greater number of epitopes.

Improvements of the acellular vaccines could also be achieved by using novel adjuvants for pertussis. Combination of aP vaccine with adjuvants that are able to drive Th1 and Th17 responses would be expected to enhance protection. Cyclic di-GMP, MF59 emulsions, the combination of aluminium hydroxide with the TLR-4 agonist monophosphoryl lipid A, have been shown to enhance Th1 type immune responses however the impact in protection of these adjuvants was not deeply investigated (Geurtsen et al. 2007; Allen et al. 2018). The B. pertussis lipoprotein BP1569, a TLR-2 agonist that activates murine dendritic cells and macrophages has recently been shown to possess adjuvant properties (Dunne et al. 2015). Recently it was reported that this protein in combination with c-di-GMP synergistically induces the production of IFN-β, IL-12 and IL-23, and maturation of dendritic cells (Allen et al. 2018). Parenteral immunization of mice with an experimental aP vaccine formulated with this combined adjuvant promoted Th1 and Th17 responses and conferred protection against lung infection with B. pertussis. Interestingly, intranasal immunization with this vaccine induced potent B. pertussis-specific Th17 responses and IL-17-secreting respiratory tissue-resident memory (TRM) CD4 T cells, and conferred a high level of protection against nasal colonization (sterilizing immunity) as well as lung infection. Furthermore, long-term protection against nasal colonization with B. pertussis was observed. This formulation would thus prolong the duration of the protective response but it is not clear that it is capable of overcoming the deficiencies of the current acellular vaccines against the circulating bacterial population. More research must be done in this regard.

7 Outer Membrane Vesicles as Vaccine Candidates Against B. pertussis Infections

All Gram-negative bacteria that have been investigated so far are able to naturally release spherical structures originated from the outer membrane (referred to as outer membrane vesicles, OMVs). Although OMVs formation seems to be a common feature of Gram-negative bacteria, the knowledge of their biogenesis and biological roles remains limited. OMVs naturally contain multiple native surface-exposed antigens as well as immunostimulatory molecules. Based on their aforementioned immunogenic potency and on positive examples of the OMV-derived vaccines against Neisseria meningitides serogroup B, we initiated several studies over the last years to analyze the potential of OMVs derived from Bordetella pertussis as vaccine candidates (Hozbor et al. 1999; Roberts et al. 2008; Asensio et al. 2011). We characterized the composition of the pertussis nanoparticles at >200 protein components—including the virulence factors PT, PRN, fimbriae, FHA, and adenylate-cyclase (Hozbor 2016). The presence of a high number of immunogens in the vaccine formulation is essential since they may avoid the high selective pressure conferred by a single or a few protective-vaccine antigens. To date, we have obtained almost 50 batches of B. pertussis–derived OMVs with robust results. Our OMV-based vaccine is safe and exhibits an adequate protection capacity against different B. pertussis genetic backgrounds, including those not expressing the vaccine antigen PRN (Gaillard et al. 2014).

The OMVs derived from B. pertussis represent an attractive acellular pertussis vaccine candidate (Hozbor 2016; Ormazabal et al. 2014; Asensio et al. 2011; Roberts et al. 2008) not only because of its safety and ability to induce protective Th1, Th17 cells (Mills et al. 1993; Ryan et al. 1997; Raeven et al. 2014; Warfel and Merkel 2013; Ross et al. 2013) and TRM cells, but because it contains a greater number of immunogens in conformations close to those found in pathogen, when compared with the current aP vaccines (Hozbor 2016; Advani et al. 2011). Consistent with previous reports (Hegerle et al. 2014; Safarchi et al. 2015), we found that immunization with commercial aP vaccine does not protect against PRN deficient isolate as effectively as against B. pertussis Tohama strain (PRN+). Since the PRN deficient isolate is not isogenic to B. pertussis Tohama strain (PRN+) and contains polymorphisms at other loci that may affect the fitness of these bacteria, we have also examined the protection of the OMV based vaccine against a PRN defective mutant derived from B. pertussis Tohama strain. We found that the commercial aP vaccine but not the OMV based vaccine exhibits lower level of protection against the PRN deficient strain when compared with the parental PRN(+) positive strain. These results clearly showed the impact of the absence of PRN expression in the effectiveness of aP vaccine against B. pertussis when comparisons are made on strains that contain the same genetic background (submitted manuscript).

The results obtained here clearly showed that the OMVs vaccine is more effective than a current commercial aP vaccine against PRN deficient strains. Therefore, the OMV formulation appears as an attractive vaccine candidate that could replace the current aP without causing concern on the reactogenicity associated with wP vaccines because of the proven safety of the OMVs vaccines (Bottero et al. 2016). Since major limitations of the current aP are their strong selection pressure exerted on the circulating bacterial population and their failure to induce sustained protective immunity, the OMV-based vaccine, that contains high number of antigens and that induces INF-γ and IL17-secreting TRM cells, has the potential to replace the current aP vaccine.