What Is Wrong with Pertussis Vaccine Immunity?

Abstract

Pertussis is resurgent in some countries, particularly those in which children receive acellular pertussis (aP) vaccines in early infancy and boosters later in life. Immunologic studies show that, whereas whole-cell pertussis (wP) vaccines orient the immune system toward Th1/Th17 responses, acellular pertussis vaccines orient toward Th1/Th2 responses. Although aP vaccines do provide protection during the first years of life, the change in T-cell priming results in waning effectiveness of aP as early as 2–3 years post-boosters. Although other factors, such as increased virulence of pertussis strains, better diagnosis, and better surveillance may play a role, the increase in pertussis appears to be the result of waning immunity. In addition, studies in baboon models, requiring confirmation in humans, show that aP is less able to prevent nasopharyngeal colonization of Bordetella pertussis than wP or natural infection.

Acellular pertussis vaccines provide protection during the first years of life, but their effectiveness wanes a few years post-boosters. This may be because these vaccines tend to produce Th1/Th2 responses (instead of Th1/Th17).

Great Debates

What are the most interesting topics likely to come up over dinner or drinks with your colleagues? Or, more importantly, what are the topics that don't come up because they are a little too controversial? In Immune Memory and Vaccines: Great Debates, Editors Rafi Ahmed and Shane Crotty have put together a collection of articles on such questions, written by thought leaders in these fields, with the freedom to talk about the issues as they see fit. This short, innovative format aims to bring a fresh perspective by encouraging authors to be opinionated, focus on what is most interesting and current, and avoid restating introductory material covered in many other reviews.

The Editors posed 13 interesting questions critical for our understanding of vaccines and immune memory to a broad group of experts in the field. In each case, several different perspectives are provided. Note that while each author knew that there were additional scientists addressing the same question, they did not know who these authors were, which ensured the independence of the opinions and perspectives expressed in each article. Our hope is that readers enjoy these articles and that they trigger many more conversations on these important topics.

Pertussis is a respiratory disease caused by several species of Bordetella, particularly B. pertussis. Inactivated and alum-adjuvanted whole-cell vaccines against pertussis were developed early in the 20th century and shown to have reasonable efficacy, at least in children. However, the specifics of whole-cell vaccines differed from manufacturer to manufacturer, resulting in variable reactogenicity and efficacy. A pooled estimate of efficacy of whole-cell vaccines gave an estimate of 78% with a wide range, but was flawed by differences in exposure (Jefferson et al. 2003).

Dissatisfaction with reactions to wP vaccines, real or perceived, led to their replacement by aP vaccines at the end of the 20th century. In the 1990s, many safety and efficacy studies of the new vaccines were conducted, but interpretation of the results was clouded by three problems: the differences between the whole-cell vaccines used as comparators, the varying concentrations of antigens contained in the candidate acellular vaccines, and the absence, for the most part, of head-to-head comparisons among the many acellular vaccines tested. Expert opinions about the results were not uniform but all observers concluded that aP vaccines are less reactogenic and that no aP vaccine was clearly superior in efficacy to a good whole-cell vaccine, whereas some asserted that multiple pertussis antigen acellular vaccines were superior in efficacy to those containing one or two antigens (Greco 1996; Gustafsson 1996; Olin 1997).

Acellular pertussis vaccines were adopted by the United States and many European countries later in the 1990s. Japan had adopted acellular vaccines even earlier in the 1980s. In subsequent years, the better tolerability of acellular vaccines was confirmed so, in that respect, the new vaccines were successful, but then the incidence of pertussis seemed to increase in the face of vaccination both in vaccinated and unvaccinated school children and adolescents. Although other possible explanations were proposed (Kilgore et al. 2016; Sealey et al. 2016), the resurgence of pertussis infections in several countries with high vaccination coverage raised questions about the nature and durability of vaccine-induced immunity (Domenech de Celles et al. 2016; Locht 2016). As reviewed below, epidemiological studies showed that the new vaccines were indeed protective in the first years after vaccination (Bisgard et al. 2005) but that protection was temporary, with rapid waning as years passed postvaccination (Plotkin et al. 2014a,b). While duration of immunity after wP has not been directly compared to that after aP, old studies suggest longer effectiveness of the former (Wendelboe et al. 2005). To complicate matters, studies in the baboon model of pertussis (also described below) suggested that acellular vaccines do not prevent colonization by the pertussis organism or its transmission to other animals. Both of these phenomena decrease the value of acellular vaccines and require immunologic explanations to improve the vaccines now in use. It should be added that a return to whole-cell vaccines will not be acceptable to the countries now using aP and other solutions must be found.

IMMUNE PROTECTION BY NATURAL INFECTIONS

Immunity to B. pertussis has not been extensively studied over the past decades. Some recent studies are shedding light however on the innate and adaptive immune arms involved in pertussis infections (Brummelman et al. 2015b; Higgs et al. 2012). The actual duration of protection conferred by natural infection in humans is not fully established, but is thought to range between 10 and 20 years. Duration of protection varies among individuals but also among studies, partly because of varying rates of asymptomatic exposures to the natural pathogen (Domenech de Celles et al. 2016; Wendelboe et al. 2005). While no studies have been designed to specifically address this question, laboratory-confirmed cases of reinfection have clearly been documented, suggesting that natural infections do not completely protect people from being reinfected 10–15 years post–primary infection (Wirsing von Konig et al. 1995; Versteegh et al. 2002). The number of well-documented cases of recurrent disease is limited, indicating that natural immunity is nonetheless rather robust. Vaccines that can achieve similar immunity to that induced by natural infections should be more than satisfactory and better than what current primary and booster pertussis vaccines can provide for long-term protection (Cherry 2014).

VACCINES: FROM WHOLE-CELL TO SUBUNIT VACCINES

The introduction of wP vaccines in the 1940s with gradual extension to most countries drastically reduced the occurrence of whooping cough by 99%. Because of significant side effects such as low grade fewer, local reactions, and some more severe rare adverse events subunit acellular vaccines based on one to five Bordetella antigens (Ags) (pertussis toxin [PT], pertactin [PRN], filamentous hemagglutinin [FHA] fimbriae 2 and 3) were developed in the early 1990s, first used as toddler and school-entry boosters and then several years later introduced as well for primary infant vaccination (Decker et al. 1995; Edwards et al. 1995; Pichichero et al. 1997; Sheridan et al. 2014; World Health Organization 2015). Whole-cell pertussis vaccines varied in quality, but the best wP vaccines gave very good protection (Plotkin 1997; Plotkin and Cadoz 1997), and the antibody levels they induced were efficiently boosted by aP vaccines (Pichichero et al. 2005, 2006). After wP priming, memory B cells persist longer than circulating antibody (Hendrikx et al. 2011b). In contrast, pertussis-specific T-cell responses do not persist nearly as well after aP priming (Palazzo et al. 2016).

THE DURATION OF EFFECTIVENESS PROBLEM: WANING IMMUNITY

The conversion from wP to aP solved the reactogenicity problem, but contributed to another: faster waning of immunity in adolescents given aP booster vaccines after aP priming, contributing to the resurgence of pertussis and to the perception that aP vaccines are insufficiently effective. Resurgence of pertussis, predicted by Hewlett and Edwards (2005), may not be due entirely to waning immunity. There have been genetic changes in B. pertussis (Cherry 2012), improved diagnostics and differing case definitions, additional public health surveillance, and collections of unvaccinated populations, although these factors will not be discussed in detail here. Among the many proposed factors leading to resurgence, the striking decline in effectiveness over time after “distant” booster vaccination forces us to focus on immunologic memory.

Initial efficacy studies for both wP and aP were conducted over short periods of time and showed that aP vaccines were indeed protective. Investigators in Europe and Senegal worked to determine estimates of long-term efficacy. These studies of children who received a three- or four-dose series suggested that protection waned faster after aP than after wP (Salmaso et al. 2001; Lugauer et al. 2002; Lacombe et al. 2004).

After both a three-dose and five-dose primary series of aP, protection predictably wanes after the last dose of the series, with the odds of pertussis increasing by 1.33 times (95% CI 1.23–1.43) for every year after receipt of diphtheria, tetanus, and acellular pertussis (DTaP)⁴ (McGirr and Fisman 2015). Epidemiological studies confirm that protection, whereas robust at time of vaccination, is temporary with waning immunity as years pass postvaccination (Table 1, Fig. 1). Waning immunity after aP was suggested early after its introduction (Lugauer et al. 2002; Lacombe et al. 2004). It became evident in the 2010 California epidemic, in which infants who could not be vaccinated because of young age were at highest risk of infection. Also striking was that 66% of cases in fully vaccinated children were in the 7- to 10-year-old age group, who had previously received only aP, suggestive of waning immunity (Winter et al. 2012). In Spain, where aP was not adopted until 2005, the incidence rate of disease significantly increased in 2010–2012 in all age groups, including unvaccinated infants <3 mo of age (Sizaire et al. 2014). Taken together, those epidemiology studies indicate that an aP primary series—when compared to a wP primary series—elicits an insufficient priming to ensure optimal boosting and long-term protection, especially in response to the later aP boosters in adolescents.

Table 1.

Studies demonstrating waning effectiveness

Year location/reference	Study design	Patient population	Vaccine	Statistical measure	Results
2002–2010, Minnesota, United States (Tartof et al. 2013)	Cohort	224,378 children: 458 cases	Five doses DTaP	Risk ratio reference: year 1 after vaccination series	Year after fifth dose: RR (95% CI) 2: 1.9 (1.3–2.9) 3: 2.6 (1.7–3.8) 4: 3.2 (2.1–4.8) 5: 6.1 (4.1–8.9) 6: 8.9 (6.0–13.0)
2002–2010, Oregon, United States (Tartof et al. 2013)	Cohort	179,011 children: 89 cases	Five doses DTaP	Risk ratio reference: year 1 after vaccination series	Year after fifth dose: RR (95% CI) 2: 1.3 (0.6–2.8) 3: 1.5 (0.7–3.7) 4: 1.7 (0.8–3.7) 5: 2.6 (1.2–5.6) 6: 4.0 (1.9–8.4)
2005–2009, Australia (Quinn et al. 2014)	Matched case-control	3123 cases, 61,636 controls	Three doses DTaP	(1-OR) × 100 reference: unvaccinated patients	Age in years: VE% (95% CI) 1: 79.2 (75.0–82.8) 2: 70.7 (64.5–75.8) 3: 59.2 (51.0–66.0)
2006–2011, California, United States (Klein et al. 2012)	Case-control	277 cases, matched controls	Five doses of DTaP	Odds ratio reference: PCR-negative controls	Odds ratio (95% CI) per year after fifth DTaP dose, 1.42 (1.21–1.66)
2006–2015, California, United States (Klein et al. 2016)	Cohort	279,493 children: 1207 cases	DTaP primary series +1 Tdap	(1-HR) × 100 reference: Tdap-unvaccinated patients	Years post-Tdap: VE% (95% CI) ≤1: 68.8 (59.7–75.9) 2: 56.9 (41.3–68.4) 3: 25.2 (-4.3–46.4) 4+: 8.9 (-30.6–36.4)
2010, Marin County, CA, United States (Witt et al. 2012)	Population-based cohort	22,798 children: 132 cases	aP	Screening method 1-[PCV/(1-PCV)] × [(1-PPV)/PPV]	Age in years: VE% (95% CI) 2–7: 41 (21–54) 8–12: 24 (0–40) 13–18: 79 (73–84)
2010, California, United States (Misegades et al. 2012)	Case-control	682 cases, 2016 controls	Five doses DTaP	(1-OR) × 100 reference: unvaccinated patients	Months since fifth dose: VE% (95% CI) <12: 98.1 (96.1–99.1) 12–23: 95.3 (91.2–97.5) 24–35: 92.3 (86.6–95.5) 36–47: 87.3 (76.2–93.2) 48–59: 82.8 (68.7–90.6) 60–83: 71.2 (45.8–84.8)
2012, Washington, United States (Acosta et al. 2015)	Case-control	Adolescents: 450 cases, 1246 controls	Tdap	(1-OR) × 100 reference: Tdap-unvaccinated patients	Months postvaccination: VE% (95% CI) <12: 73.1(60.3–81.8) 12–23: 54.9 (32.4–70.0) 24–47: 34.2 (-0.03–58.0%) Overall: 63.9 (50–74)
2012, Wisconsin, United States (Koepke et al. 2014)	Cohort	Adolescents: 940 cases matched to Tdap-vaccinated controls	Tdap	(1-IRR) × 100 reference: Tdap-unvaccinated patients	Year postvaccination: VE% (95% CI), baseline:<1: 75.3 (55.2–86.5) 1: 68.2 (60.9–74.1) 2: 34.5 (19.9–46.4) 3–4: 11.9 (-11.1–30.1)
2012, Oregon, United States (Liko et al. 2014)	Population-based cohort	709 cases ages 2 mo–19 years partially or fully vaccinated	aP	[1-(AR vaccinated/AR unvaccinated)] × 100	Age: VE% (95% CI) 15–47 mo old: 95 (92–97) 4–6 yo: 89 (81–94) 7–10 yo: 83 (72–90) 11–12 yo: 65 (46–78) 13–16 yo: 47 (19–65) 17–19 yo: 66 (30–84)

AR, Attack rate; HR, hazard ratio; IRR, incidence rate ratio; OR, odds ratio; PCR: polymerase chain reaction, PCV, proportion of cases vaccinated; PPV, proportion of population vaccinated; RR, relative risk.

Figure 1.

(A) Demonstration of waning immunity following either infant/early childhood diphtheria, tetanus, and acellular pertussis (DTaP) or early adolescent tetanus, reduced diphtheria, and acellular pertussis (TdaP), measured as vaccine effectiveness. Vaccine effectiveness was calculated in each study as described in Table 1. The California five-dose study refers to Misegades et al. (2012), whereas the TdaP study refers to Klein et al. (2016). *Last point is 24–47 mo post-booster. (B) Demonstration of waning immunity with DTaP measured as relative risk (RR) of infection, with regard to first-year post-booster vaccination.

ACELLULAR VACCINES: HOW DO THEY PROTECT COMPARED TO KILLED wP VACCINES?

Thus, clinical and epidemiology results showed that protection induced by aP vaccines was potent and quite similar or better than the one achieved with wP (Edwards and Decker 2013). The improved safety profile led to the decision to switch to aP vaccines alone in most developed countries initiated in the mid-1990s (Klein 2014). A review of effectiveness and efficacy studies of wP and currently available aP vaccines suggested that even after the primary series, aP vaccines are less efficacious (84%) than wP vaccines are effective (94%) (Fulton et al. 2016). However, the heterogeneity of whole-cell vaccines and the paucity of clinical trials using them make conclusions difficult.

Studies in Australia and the United States suggested that patients primed with wP have longer-lasting protection than those who had aP, although because of variability in wP it is unknown whether that is also the case in countries with a less efficacious wP vaccine (Sheridan et al. 2012, 2014; Klein et al. 2013; Liko et al. 2013; Witt et al. 2013). Even with a five-dose DTaP series and Tdap⁵ booster, adolescents immunized in infancy with DTaP instead of at least the first dose of the primary series having been wP vaccine had an increased incidence of pertussis disease (Liko et al. 2013; Skoff and Martin 2016).

The main differences identified in humans between immune responses induced by aP vaccines compared to those triggered by wP or natural infections are detailed in Table 2. Those results remain still rather limited for vaccines having been licensed for decades and can even be sometimes a bit contradictory. Some overall trends nonetheless emerge but should be analyzed with caution and require further investigation.

Table 2.

Immunity to B. pertussis infection and vaccines in humans

Immune responses to:	Humoral responses		T-cell responses
	Antibody (Ab) levels	Memory B cells	Th	Memory T cell
Natural infections	Levels and avidity index of anti-PT antibodies is higher in infected individuals compared to pertussis (wP/aP)- vaccinated individuals (Barkoff et al. 2012); Abs recognizing protective PT epitopes better elicited by natural infections compared to aP (Sutherland et al. 2011)	Pertussis-specific IgG levels and memory B cells decrease with time postinfection (9 mo); overall weak correlation between circulating pertussis IgG response and memory B cell frequency; PT responses decline faster than FHA and PRN ones; higher numbers of pertussis memory B cells in older (adult and elderly) compared to younger age (4 yr and preschool) in the acute phase (van Twillert et al. 2014); lower memory B cells and Abs responses in infants undergoing recurrent episodes of acute otitis media (Basha and Pichichero 2015)	Lower magnitude of PT-specific T-cell responses in naturally infected children (8–59 mo) compared to aP-vaccinated kids (Ausiello et al. 2000); Th1/Th17 responses are elicited (Mascart et al. 2003; Schure et al. 2012a)
Whole-cell pertussis vaccines	Lower Abs levels and avidity with wP compared with aP at 4 yr of age after primary series; preschool booster induces stronger responses in wP primed (Schure et al. 2013); avidity of Ab levels upon aP boost can be increased similarly in wP-primed and aP-primed adolescents (Prelog et al. 2013)	Lower frequency of memory B cells compared to aP (4 yr of age) after primary series; preschool booster induce stronger memory B-cell responses in wP primed (Schure et al. 2013)	Lower IFN-γ secreting T cells when compared to aP primary series (4 yr of age); preschool booster induces stronger Th responses in wP primed (Schure et al. 2013)	Better preschool boostability in wP-primed versus aP-primed individuals at 4 yr of age (Schure et al. 2012b)
Acellular pertussis	Th2 skewing (IgG4) after primary aP series in infants (Hendrikx et al. 2011c) and in adolescents (Jahnmatz et al. 2014b); lower avidity of PT-specific Ab responses when compared to those upon natural infections (Barkoff et al. 2012)	80% of aP-primed infants still show circulating memory B cells after primary series (Carollo et al. 2014); aP booster vaccine improve B-cell memory immunity (> wP) in 4-yr-old children in some cases but it wanes gradually (Hendrikx et al. 2011a)	aP booster vaccines improve T-cell (helper and memory) immunity (> wP) in 4-yr-old and 9-yr-old children (Schure et al. 2012b); Th1 in adolescents upon booster aP series, irrespective of wP or aP primary series (Rieber et al. 2011); overall, aP induces a more Th2 or balanced Th1/Th2 profile (Ausiello et al. 1997; Ryan et al. 1998; White et al. 2010); Th1 responses impaired upon vaccination during pregnancy (Huygen et al. 2015); T-cell immunodominance patterns are similar for wP and aP; the respective Th1 and Th2 skewing of wP and aP are unchanged for decades with aP boosters (Bancroft et al. 2016)	As good CD4 and CD8 T cells priming between aP and wP, there is a limited effect of a fifth aP dose in preschool children (de Rond et al. 2015); level of functionality (cytokine production) is weaker in aP-vaccinated preadolescents (Smits et al. 2013); 10 years post–last booster, T-cell responses still detectable even after Abs have decayed (Grondahl-Yli-Hannuksela et al. 2016)

IMMUNE RESPONSES TO PERTUSSIS VACCINES IN HUMANS

Even if no correlates of protection have been widely accepted for pertussis vaccines, it is well documented that Ab responses are critical effectors to mediate protection. PT antibodies are particularly correlated with protection (Plotkin 2013). On a wP priming background, aP boosters induce circulating Ab levels that rapidly increase and decrease within months, but remain well above pre-booster baselines for years (Baretto et al. 2007; Dalby et al. 2010), and are well recalled on additional boosting (Pichichero et al. 2005, 2006; Baretto et al. 2007; Halperin et al. 2012). Antibody and B-cell responses elicited by aP vaccines seem to be close to those induced by wP and natural infection, even if some studies support stronger booster effects in wP-primed individuals (Schure et al. 2013). The rapid decay of Ab responses seems thus to be related to specific attributes of Bordetella antigens rather than to the use of a subunit vaccine and could be partly explained by an intrinsic poor priming property of B. pertussis–specific B cells on antigen/pathogen encounter (van Twillert et al. 2015). In addition, like many pathogens causing persistent infections, B. pertussis has developed several mechanisms to subvert host immunity, such as defects in memory T–cell homing (Nguyen et al. 2012), and induction of regulatory T cells (McGuirk et al. 2002; Ross et al. 2004, 2013; Coleman et al. 2012) that may negatively affect immune priming, including humoral responses.

Even less information is known about pertussis-specific T-cell responses elicited by vaccines. This has only been more recently investigated because of the resurgence of infections suggested to be partly associated with defects in T-cell immunity. The limited set of data indicates that aP vaccine induces Th2-skewed or mixed Th1/Th2 responses, whereas wP vaccines and natural infections skew T-cell responses toward a more Th1 profile (Mascart et al. 2003; Dirix et al. 2009; Higgs et al. 2012; Vermeulen et al. 2013; Edwards and Berbers 2014). Priming with wP (recalled with aP boosters) gives rise to a stronger Th1 response in adolescents than an aP priming (Smits et al. 2013). This has been recently confirmed by a study showing a transient increase in “bystander” IgE responses in infants and preschoolers having received only aP-containing vaccines versus those having been primed with wP (Holt et al. 2016). Very little was known of Th17 cells until rather recently, but two studies report that aP vaccines fail to elicit Th17 responses (Schure et al. 2012b, 2013). Additional findings in vitro on human cells may explain why aP, with its more restricted Ag composition and per the nature of the alum adjuvant used, is not as well equipped as wP and natural infection to trigger Th17 responses (Fedele et al. 2008, 2010; Dunne et al. 2015). Beyond this difference in T-helper polarization, few if any quantitative differences have been identified in humans between wP and aP immunizations: similar frequencies and equivalent persistence, if not more, of memory and helper T cells have been observed for wP and aP vaccines (Table 2). Similarly to humoral immunity, memory T–cell responses can be sustained for years by aP boosters with little overt waning.

ANIMAL MODELS

To compensate for the limited inferences from clinical results, immune responses to pertussis vaccines have been more thoroughly deciphered in animal models. On top of a hypothetically weaker B-cell priming observed in humans, one mouse study provides possible mechanistic insights into the waning of humoral responses (Stenger et al. 2010). Three limitations in the long-term maintenance and function of pertussis memory Ab responses after aP were proposed: a more limited self-renewal capacity of memory B cells, a defect in the differentiation ratio between plasma cells and memory B cells from germinal center B cells, and an exhaustion of plasma cell in the bone marrow on accumulation of boosts. No other studies have, however, confirmed these hypotheses. Interestingly, it has also been described in mice that B. pertussis–specific memory B cells provide direct protection in the absence of circulating antibodies (Leef et al. 2000; Mahon et al. 2000), indicating their key role in protection provided they can support a functional booster response in due time before pertussis has time to develop.

Animal models have been very valuable to better understand the underlying mechanisms of the defect in T-cell skewing associated with aP vaccinations. In mice, it has been shown that aP vaccines differ from both wP vaccines and natural infection in terms of Th polarization. First, as observed in humans, aP induces a more balanced Th1/Th2 profile when compared to either wP or natural infection, which both direct a more Th1-skewed response (Mills et al. 1993, 1998; Brady et al. 1998; Sugai et al. 2005; Polewicz et al. 2013). Second, aP fails to elicit Th17-specific cells, whereas wP can better mimic natural infection by the induction of this particular subset (Higgs et al. 2012; Brummelman et al. 2015b; Dunne et al. 2015). More importantly, a recently developed baboon model nicely recapitulates pertussis disease and has become instrumental to investigating Bordetella immunity and to compare the different vaccines and infection (Warfel and Merkel 2014). This model is recognized as well suited to study pertussis vaccine performance for many reasons: it permits physiologically relevant intranasal challenges including in infant animals; the baboon immune system is close to the human one, including the same four IgG isotypes; and serial samplings including mucosal ones can be performed to track infection and precisely monitor protection. In baboons, aP vaccines protect adolescent primates as well as wP or natural infection against disease (no symptoms) but fail to prevent infection (colonization) and thus transmission (Warfel et al. 2014b). No major differences in Ab titers against the vaccine Ags were observed between aP and wP vaccines, indicating an important role of T cells in protection against infection. Accordingly, wP-vaccinated animals show a Th1/Th17 skewing similar (although milder) than the one induced by natural infection, whereas aP-vaccinated baboons show balanced Th1/Th2 responses, with no signs of Th17 immunity. In an infant challenge model, both maternal and neonatal vaccinations have been assessed (Warfel et al. 2014a). Although the study is limited by the number of animals used, maternal immunization with aP-induced vaccine-specific titers in neonates are sufficient to confer full protection against disease even if all were colonized. Similarly, aP vaccination of neonates (2 days old) induced significant antibody responses (that could be further boosted by an immunization at day 28), which fully protected baboons from disease even if they remained heavily colonized. These animal results suggest that the T-cell profile induced by aP vaccines is inefficient at conferring protection against colonization and hence transmission, and thus induction of a different T-cell response may be crucial to more prolonged protection.

HERD IMMUNITY

The ability of wP to block transmission in humans remains a matter of debate: the Fine and Clarkson (1982) study failed to show prevention of transmission, whereas other studies have established strong signatures of herd immunity on mass vaccination (Preziosi and Halloran 2003; Wearing and Rohani 2009; Blackwood et al. 2013). One recent study in humans indicate that siblings are now the most common source of transmission to infants, although direct studies of colonization in humans are needed (Skoff et al. 2015). A recent public health investigation in Florida suggested that there is sustained transmission of B. pertussis in an aP-vaccinated cohort (Matthias et al. 2016). Not only, then, do aP-vaccinated children have waning immunity, but even those with immunity can contribute to ongoing circulation of disease. While it is difficult to measure asymptomatic carriage, Althouse and Scarpino have designed mathematical models based on the genetic analysis of B. pertussis isolates in the United States and United Kingdom, which suggests that some of the resurgence of B. pertussis infections can be explained by asymptomatic transmission (Althouse and Scarpino 2015). The possibility of a lower ability of aP to limit B. pertussis colonization is a key parameter to take into account when considering design of a more potent subunit pertussis vaccine.

EVOLUTION OF B. pertussis

Strain change as a mechanism has been proposed leading to resurgence (Mooi et al. 2014). For example, a recent genomic analysis of strains in an outbreak in the United Kingdom found that aP vaccine antigen-encoding genes evolved at a high rate than other surface protein-encoding genes, supporting the hypothesis that aP immunity may drive the evolution of B. pertussis (Sealey et al. 2015, 2016). Reassuringly, whereas pertactin-deficient strains are increasing in the United States, vaccine effectiveness was maintained in a setting of high (>90%) pertactin-deficient circulating strains (Breakwell et al. 2016). Austrian investigators have identified more severe disease in infants infected with a polymorphism of the pertussis toxin operon, ptxP3, which results in increased toxin production. In a mouse model, Australian strains carrying the ptxP3 promoter colonized more efficiently than older strains (Safarchi et al. 2016). Other vaccine components may be more critical in preventing disease and additional studies examining the immune response to filamentous hemagglutinin or fimbrial antigens need to be conducted. In addition, strains with the ptxP3 promoter are more common in countries using aP than countries using wP, which provides another reason why pertussis has increased more in the former (He 2016).

WHAT NEEDS TO BE DONE TO BETTER UNDERSTAND CURRENT SHORTCOMINGS OF THE aP LONG-TERM IMMUNITY?

To have sustainable humoral responses with fewer boosters, a much more in-depth analysis of the induction, maintenance, and function of the plasma cell and memory B–cell pools is absolutely required to better understand what immune events limit the mobilization and amplification of memory arms. Similarly, more baboon and mouse studies are needed to confirm and better comprehend the differential Th skewing triggered by aP compared to natural infections or wP immunizations. Identifying the pathways and biomarkers that underpin those differences will be critical for the development of improved aP primary series vaccines. Those studies should exploit all the most recent technologies of system biology (transcriptomics, metabolomics, proteomics …) for an in-depth differential characterization of the features of both vaccine types. First attempts to tackle this biomarker quest using Omics approaches have been initiated and some immune signatures preceding the generation of Th1 and Th17 cells and IgA in the lungs after natural infections were shown in mice (Raeven et al. 2014). Similar studies should be performed in humans either primed with aP or wP vaccines, aiming to profile the different immune signatures imprinted during primary series, to confirm animal results and hopefully to corroborate preclinically identified biomarkers of aP defects.

It is of upmost importance to determine the magnitude and the nature (Th skewing) of sustained functional humoral and cellular memories that are required to support protection; this will establish the main immunologic criteria against which new aP vaccines can be selected. In addition, we do not know how T follicular helper cells (Tfh) are regulated during pertussis infections or immunizations, although these cells are known to be instrumental in orchestrating humoral memory immunity (Hale and Ahmed 2015).

WHAT ARE THE OPTIONS TO IMPROVE CURRENT aP VACCINES?

We must stress that aP vaccines remain very potent and safe vaccines that generally protect worldwide populations against B. pertussis disease. The short to mid-term protection is equal to that induced by wP pertussis. Recent resurgences and outbreaks have raised questions about the ability of aP priming to optimally support longer term protection, leading to the characterization of a different immune profile between aP and wP vaccines. However, this is definitely not the only causative factor to consider, as resurgences have also occurred in countries still using wP vaccines (Tan et al. 2015). Even if the underlying immune mechanisms of the long-term aP “weaknesses” are not yet fully elucidated, more Th2 skewed immune responses, some defects in memory T– and B–cell functions, and clinical and animal data jointly show that aP vaccine priming appears to be less optimal than after wP vaccines and natural infections. This is especially true for their ability to sustain long-term protection and fully efficient boostability (Bolotin et al. 2015).

Considering the good ability of aP to boost existing responses even if, for a short period of time, a less disruptive option could be to test new vaccine schedules in infants/adolescent to try to prevent waning of immunity (Sharma and Pichichero 2012). Priming with aP in infants is not efficient, but after the age of 5 (once immune maturity is fully reached) boosting is quite optimal (van Twillert et al. 2014) and more boosters could possibly fix the current situation (Gabutti et al. 2015). Change in infant vaccination however is not an easy task regulatory wise, and this option is probably not the most appropriate to make significant immune improvements. Vaccination of individuals having contact with newborns, often referred to as the “cocooning” strategy, and during pregnancy using current aP vaccines also are alternative options that when combined could have an impact to reduce resurgences and outbreaks (Plotkin 2014b).

The most reliable although more long-term option is to develop an improved aP vaccine to address its current shortcomings, using novel adjuvants or additional antigens, for long-term protection and prevention of colonization. This is the focus of many current efforts conducted by the public and private pertussis communities, as summarized in Brummelman et al. (2015b), Meade et al. (2014), and Plotkin (2014a) and shown in Figure 2 and noted in Table 3.

Figure 2.

New aP vaccine should be aimed to increase priming and boostability of current subunit vaccine through the addition of adjuvants that will program a more Th1/Th17 immune profile and possibly the addition of more subunit Ags inducing bactericidal activity to limit or prevent colonization. Another option, not mutually exclusive, is to deliver the vaccine and/or induce a stronger immune response to a more physiologically relevant compartment for the disease (i.e., the lung) to trigger more potent mucosal responses with hopefully a better duration as well through a better homing of memory cells in the lung mucosa. Several Ags, adjuvants, and/or routes of immunizations harboring the above properties have already been reported in the literature.

Table 3.

Possible improvements of acellular pertussis vaccines

Options (not mutually exclusive)	Examples already reported
Enhances immune priming and favors a Th1/Th17 skewing with adjuvants	CpG (Sugai et al. 2005; Garlapati et al. 2011; Asokanathan et al. 2013; Polewicz et al. 2013; Ross et al. 2013)
	TLR4 agonist (Brummelman et al. 2015a, 2016)
	cdiGMP (Elahi et al. 2014)
	TLR-2 targeting lipoprotein (Dunne et al. 2015)
Decrease colonization to reduce transmission using Ags that induce bacterial killing	Adenylate cyclase (Cheung et al. 2006; Sebo et al. 2014)
	OMV (Roberts et al. 2008; Asensio et al. 2011)
Increase lung memory immunity	By intranasal route (Fedele et al. 2011; Skerry and Mahon 2011; Feunou et al. 2014; Jahnmatz et al. 2014a; Thorstensson et al. 2014)
	By intradermal route using patch (Halperin et al. 2002), DBV Technologies/BioNet-Asia/University of Geneva (Gavillet et al. 2015)

CONCLUSIONS

The phenomena of waning immunity after boosting on an aP background, inability to eliminate colonization, and evolution of B. pertussis require the improvement of aP vaccines. It should be added that a return to whole-cell vaccines will not be acceptable to the public and other solutions must be found. The improvement of aP is essentially an immunologic problem. In a nutshell, the two principal issues are to prolong B-cell effector memory so that antibody, particularly against PT, persists for a longer period of time, and to generate a Th1/Th17 T-cell environment early in life so that carriage of Bordetella is prevented. Both goals will require reformulation of pertussis vaccine constituents, both antigens and adjuvants. The most obvious strategies are to use genetically rather than chemically inactivated PT (Seubert et al. 2014); to use adjuvants that better stimulate innate immunity and memory; and/or to add new B. pertussis virulence factors to the vaccine (Allen and Mills 2014; Rumbo and Hozbor 2014). Live pertussis vaccines are now under investigation, with possible advantages in conferring immunity as they mimic natural infection (Locht and Mielcarek 2014). Regulatory approval poses significant barriers to licensure of new vaccines, as traditional efficacy trials cannot be performed, and the appropriate antibody response to serve as a correlate of protection is disputed (Clark et al. 2012).