Innovative adjuvant strategies for next-generation pertussis vaccines

ABSTRACT

The acellular pertussis (aP) vaccine has increasingly replaced the whole-cell pertussis (wP) vaccine due to its superior safety profile. However, the aP vaccine is less effective at preventing infection and transmission of Bordetella pertussis, highlighting the need for more effective aP vaccines. Current aP vaccines do not elicit the robust cellular immunity necessary to eliminate intracellular bacteria and do not induce sufficient mucosal immunity to prevent bacterial colonization in the upper respiratory tract. Incorporating novel adjuvants represents a promising avenue for the future development of pertussis vaccines. Nevertheless, there remains a significant gap in understanding the application of novel adjuvants. In this article, we summarize the currently approved pertussis vaccines, focusing on the types of antigens and adjuvants used, and discuss the mechanisms of novel adjuvants. This provides valuable insights into the roles of adjuvants in pertussis vaccines, laying a foundation for designing next-generation pertussis vaccines with improved adjuvant systems.

Introduction

Pertussis is a severe infectious respiratory disease that affects infants, children, and adults.^1,2 The incidence of pertussis declined rapidly only after the development of whole-cell pertussis (wP) vaccine in 1948, when cases dropped from 157 cases per 100,000 in 1940 to 1 case per 100,000 in 1973.³ Although the wP vaccine is sufficiently effective, the acellular-pertussis (aP) vaccine is becoming more dominant because of its better safety record.⁴ However, the outbreak of pertussis in recent years remains a concern.⁵ Although the number of reported cases and the incidence of pertussis decreased between 2020 and 2022 due to extensive public health measures during the COVID-19 pandemic,⁶ 62646 cases were reported globally in 2022⁷ In 2023, the number of pertussis cases worldwide increased to 158,910.⁸ As of November 30, 2024, more than six times as many pertussis cases have been reported in the United States compared to the same period in 2023.⁹

There are several reasons for the resurgence of pertussis, including regional differences in vaccine coverage, the gradual decline of immunity gained through natural infection or vaccination over time, the increase in B. pertussis isolates that do not produce certain vaccine antigens (e.g., PRN), which provides a selective advantage for bacterial survival in population vaccinated with aP vaccines, the absence of virulence factors such as adenylate cyclase toxin in vaccines that are necessary to induce a protective immune response, and limitations on the immunomodulatory activity of certain antigens like filamentous hemagglutinin that promotes the production of immunosuppressive cytokines, etc. ^10–13 Evidence also shows that when aP vaccines replace wP vaccines, they do not provide optimal immune enhancement or long-term protection. This is closely related to the different immune stimulations caused by wP vaccines and aP vaccines using aluminum adjuvants.^13,14

This review article aims to thoroughly examine currently approved pertussis-based vaccines, including the types of pertussis antigens and the adjuvants used. It subsequently discusses the advancements in pertussis vaccine development, focusing on new adjuvants being developed for these vaccines. Our goal is to guide the design of new adjuvants in pertussis vaccines and lay the foundation for designing and developing of new combined pertussis vaccines.

Antigen components in the aP vaccine formulation

B. pertussis, which is a human pathogen infecting the respiratory tract, produces various virulence factors on the mucosal surface of the host¹⁵, including pertussis toxin (PT), FHA, pertactin (PRN), and fimbriaes (FIM), which come in two types: Fim2 and Fim3 (Figure 1).^16,17 These antigens are frequently included in aP vaccines, which have been found to play a crucial role in providing protection against pertussis infection.

Figure 1.

Schematic representation of B. pertussis and its virulence factors. B. pertussis produces several virulence factors, including pertussis toxin (PT), adenylate cyclase toxin (ACT), tracheal cytotoxin (TCT), dermoncrotic toxin (DNT), filamentous hemagglutinin (FHA), fimbriae (fim), pertactin (PRN), bordetella resistance killing antigen (BrkA), and tracheal colonization factor (TCF).

PT is a secreted extracellular toxin and a key virulence factor of B. pertussis, playing a central role in the pathogenesis of whooping cough.¹⁸ It is widely believed that PT is the main contributor to the clinical manifestations of B. pertussis infection. A strong correlation exists between vaccine-induced serum PT-specific IgG levels and protection against pertussis, making PT the most representative antigen among all currently used aP vaccines.^19,20 PT follows the classical bacterial toxin A/B model: the A component possesses enzymatic activity, while the B component is responsible for binding to the target cell receptor.^21,22 In murine intranasal infection models, PT specifically targets resident airway macrophages, facilitating early colonization of the respiratory tract by B. pertussis.²³ Additionally, PT inhibits neutrophil recruitment, enabling the bacterium to evade earlier immune.²⁴ Multiple studies have demonstrated that anti-PT antibodies confer strong protection against intracerebral (IC) challenge in mice.^25–27 In Denmark, a monocomponent aP vaccine containing hydrogen peroxide-inactivated PT was licensed in 1997. It has since been used for both primary infant vaccinations (DTaP-IPV) and booster doses in children (TdaP and TdaP-IPV). Despite using PT as the sole antigen, Denmark reports a pertussis burden comparable to neighboring countries employing multi-component aP vaccines.²⁸

FHA, unlike bacterial toxin PT, is primarily involved in bacterial adhesion to tracheal epithelial cells. It is a helical protein that can be released into the extracellular environment.^22,29 Various domains within FHA facilitate binding to both macrophages and respiratory epithelial cells.³⁰ Kimura et al. demonstrated that mice immunized intraperitoneally or intramuscularly with FHA developed FHA-specific antibodies and exhibited reduced bacterial colonization in the trachea and lungs.³¹ In a human study, a Japanese two-component vaccine containing PT and FHA provided better long-term protection over 3 y compared to the monocomponent PT vaccine.³² A clinical trial in Sweden evaluated the efficacy of the single-component (PT only, JNIH-7) and two-component (PT+FHA, JNIH-6) vaccines in infants aged 5–11 months. Over a 15-month follow-up starting 30 d after the second dose, culture-confirmed pertussis occurred in 27 recipients of the monocomponent vaccine and 18 recipients of the two-component vaccine. The estimated protective efficacy was 54% for the PT-only vaccine and 69% for the PT-FHA combination.^33,34 Importantly, extended follow-up showed that the inclusion of FHA significantly reduced the incidence of B. pertussis infection compared to PT alone.³⁵

PRN is a surface-associated protein that promotes adsorption to tracheal epithelial cells via its Arg-Gly-Asp motif and proline-rich domains.^36,37 In murine models, PRN contributes to pathogenesis by inhibiting neutrophil-mediated clearance³⁸ and promoting inflammation, which enhances bacterial shedding and transmission.³⁹ Immunization with PRN has been shown to suppress bacterial proliferation in the lungs and support early clearance of infection.⁴⁰

FIM are filamentous, aggregated protein structures expressed on the bacterial surface. They function as adhesins that promote bacterial adhesion to host tissues.⁴¹ Two major serotypes, FIM2 and FIM3, represent distinct pili structures.^22,42 Holuboval et al. demonstrated that the co-adhesion mediated by both FHA and FIM is essential for nasal colonization, secretion induction, shedding, and subsequent host-to-host transmission.⁴³ Enhancing FIM content in aP vaccines has been shown in mouse studies to boost protection without increasing reactogenicity.⁴⁴ In Sweden clinical trials comparing two-, three-, and five-component aP vaccines with whole-cell pertussis (wP) vaccines, the two-component vaccine exhibited significantly lower efficacy. The five-component and three-component aP vaccines, as well as the wP vaccine, showed comparable protection against culture-confirmed pertussis with at least 21 d of paroxysmal cough. However, the three-component vaccine provides relatively weaker protection against mild disease, suggesting a role for FIM in reducing infection and transmission.⁴⁵

Acellular pertussis combination vaccine

These pertussis antigens are typically combined with tetanus and diphtheria toxins for administration. The FDA-approved human combination vaccines include INFANRIX (GlaxoSmithKline Biologicals) and DAPTACEL (Sanofi Pasteur) for children, as well as BOOSTRIX (GlaxoSmithKline Biologicals) and Adacel (Sanofi Pasteur) for adolescents and adults (Table 1). The amounts of pertussis antigens and diphtheria toxoids are lower in adolescents and adults than in children, while the amount of tetanus toxoids remains similar. DAPTACEL (Sanofi Pasteur) and Adacel (Sanofi Pasteur) are five-component vaccine preparations that contain aluminum phosphate adjuvants, whereas INFANRIX (GlaxoSmithKline Biologicals) and BOOSTRIX (GlaxoSmithKline Biologicals) are three-component vaccine preparations (without type 2 and type 3 FIM). According to the summary by Susanna Esposito and Nicola Principi on the study of Greco and Gustafsson, the efficacy of both three- and five-component vaccines appears to be similar.⁴⁶ However, Carvalho et al. report that children who received the three-antigen (aP3) aP vaccine were more susceptible to pertussis than those who received the wP vaccine. Children receiving the five-antigen (aP5) aP vaccine in their primary vaccination had a similar risk of pertussis as children who received the wP vaccine.^47,48

Table 1.

FDA-approved human vaccine containing pertussis antigens.

Trade Name	Manufacturer	Adjuvant	Pertussis Antigen per Dose				Diphtheria toxoid	Tetanus toxoid	HBsAg	Poliovirus serotypes			PRP-OMPC		PRP-TT
			PT	FHA	PRN	FIM				1	2	3	PRP	OMPC	PRP	TT
INFANRIX	GlaxoSmithKline Biologicals	Aluminum hydroxide	25 µg	25 µg	8 µg		25 Lf	10 Lf
DAPTACEL	Sanofi Pasteur, Ltd	Aluminum phosphate	10 µg	5 µg	3 µg	5 µg	15 Lf	5 Lf
Pediarix	GlaxoSmithKline Biologicals	Aluminum hydroxide,Aluminum phosphate	25 µg	25 µg	8 µg		25 Lf	10 Lf	10 µg	40 DU	8 DU	32 DU
KINRIX	GlaxoSmithKline Biologicals	Aluminum hydroxide	25 µg	25 µg	8 µg		25 Lf	10 Lf		40 DU	8 DU	32 DU
Quadracel	Sanofi Pasteur, Ltd	Aluminum phosphate	20 µg	20 µg	3 µg	5µg	15 Lf	5 Lf		40 DU	8 DU	32 DU
VAXELIS	MCM Vaccine Company	Amorphous aluminum hydroxyphosphate sulfate	20 µg	20 µg	3 µg	5 µg	15 Lf	5 Lf	10 µg	29 DU	7 DU	26 DU	3 µg	50 µg
		Aluminum phosphate.
Pentacel	Sanofi Pasteur, Ltd	Aluminum phosphate	20 µg	20 µg	3 µg	5 µg	15 Lf	5 Lf		40 DU	8 DU	32 DU			10 µg	24 µg
Adacel	Sanofi Pasteur, Ltd	Aluminum phosphate	2.5 µg	5 µg	3 µg	5 µg	2 Lf	5 Lf
Boostrix	GlaxoSmithKline Biologicals	Aluminum hydroxide	8 µg	8 µg	2.5 µg		2.5 Lf	5 Lf

In addition to the traditional tetanus-diphtheria-pertussis vaccine, the combination vaccine may also include other antigens. For example, the FDA-approved vaccines KINRIX (GlaxoSmithKline Biologicals) and Quadracel (Sanofi Pasteur) are used in combination with poliovirus antigens, with aluminum hydroxide adjuvant and aluminum phosphate adjuvant, respectively. PEDIARIX (GlaxoSmithKline Biologicals) is administered in combination with hepatitis B antigen and poliovirus antigen, with diphtheria and tetanus toxoids as well as pertussis antigens (PT, FHA and PRN) adsorbed on aluminum hydroxide. The hepatitis B component is adsorbed on aluminum phosphate. To prevent invasive diseases caused by Haemophilus influenzae type B (Hib), protein-conjugated polyribosylribitol phosphate (PRP) was added to the vaccine to enhance the immunogenicity of VAXELIS (MCM Vaccine Company) and Pentacel (Sanofi Pasteur) (Table 1). PRP, the primary virulence factor for invasive Hib disease, is a highly immunogenic antigen with demonstrated good protective effects.²⁰ The covalent conjugate of PRP and the outer membrane protein complex (OMPC) of Neisseria meningitidis serogroup B, adsorbed on amorphous aluminum hydroxyphosphate sulfate, is included in VAXELIS. Hepatitis B is also adsorbed on amorphous aluminum hydroxyphosphate sulfate, while diphtheria and tetanus toxoids as well as pertussis antigens are, respectively, adsorbed on aluminum phosphate. The covalent conjugate of PRP and tetanus toxoid is included in Pentacel, along with diphtheria, tetanus toxoids and pertussis antigens adsorbed on aluminum phosphate.

Adjuvants for the acellular pertussis combination vaccine

To enhance immunogenicity, aP-based vaccines are typically formulated with adjuvants. Adjuvants are used in inactivated, subunit, and recombinant vaccines to stimulate both innate and adaptive immune responses for optimal and long-lasting immunogenicity.⁴⁹ B. pertussis was originally considered to be a specialized extracellular pathogen, and the robust humoral immunity induced by aP vaccines containing aluminum salts as adjuvants was sufficient to prevent B. pertussis. However, B. pertussis has been found to infect and survive in human alveolar macrophages and broncho-alveolar lavage cells of mice.^14,50,51 Cellular immunity may play a crucial role in protection against pertussis.⁵² Mills et al. found that CD4⁺ T cells can mediate bacterial clearance even without a detectable serum antibody response and that the primary T cells induced by natural pertussis infection were CD4⁺ T cells secreting IL-2 and IFN-γ.⁵³ IFN-γ can activate macrophages to eliminate intracellular pertussis bacteria.⁵⁴ The Th1 cell-mediated cellular immune response is crucial for protective immunity in mice against pertussis caused by respiratory infections (Figure 2). Ross et al. identified a significant number of B. pertussis-specific Th17 cells in the lungs of mice naturally infected with pertussis. They found that the adoptive transfer of B. pertussis-specific Th1 or Th17 cells effectively protected mice from B. pertussis infection, especially when these two populations were transferred together. Both Th1 and Th17 immunity contributed to the natural immunity against B. pertussis infection in mice (Figure 2). The wP vaccine also stimulated Th1 and Th17 cells, providing protective immunity in mice mediated by IFN-γ. IL-17A also plays a role, though to a lesser extent. However, IL-17A is important in the protective immunity induced by the aP vaccine by recruiting macrophages and neutrophils to enhance the elimination of pertussis in the lungs.⁵⁵ The significance of Th1 and Th17 responses was also confirmed in baboon models, where wP vaccines that stimulated Th1 and Th17 memory responses cleared the infection more rapidly. Unlike the Th2/Th17 immune response induced in mice⁵⁵, the aP vaccine that induced the Th1/Th2 response in baboons cleared the infection slowly and was ineffective at preventing B. pertussis colonization and spread in the respiratory tract.⁵⁶ This is due to the extensive antigens and potential adjuvants of the wP vaccine, while the aP vaccine is formulated with a single adjuvant and a limited set of antigens, which can stimulate different and more restricted immune responses.⁵⁷ This has been linked to relate to the resurgence of pertussis in recent years. Therefore, in addition to aluminum salt adjuvants, new adjuvants that aim to induce Th1 and Th17 responses, such as MF59, AS04, AS01, CpG ODN, Poly (I:C), TLR7 agonists, lipoproteins, liposomes, etc., also show development potential in the novel pertussis vaccine (Figure 3). Results have been obtained in preclinical studies (Table 2).

Figure 2.

Mechanistic illustration of natural immunity to B. pertussis in mice. Following the primary respiratory infection with B. pertussis, local dendritic cells (DCs) capture the B. pertussis antigen, migrate to the drainage lymph nodes, and stimulate the initial CD4⁺ T cells to differentiate into Th1 and Th17 cells under the influence of specific cytokines. IFN-γ-secreting Th1 cells activate alveolar macrophages to clear B. pertussis from the respiratory tract. IL-17 released by Th17 cells contributes to neutrophil recruitment to the lungs. Initial CD4⁺ T cells can also differentiate into effector memory T (T_EM) cells, which move to nasal tissues and lungs, where they are retained as tissue-resident memory T (T_RM) cells. During reinfection, IL-17⁺ CD4⁺ T_RM cells expand and recruit sialic acid-binding, ig-like lectin (Siglec)-F⁺ neutrophils, which have a higher potential for forming neutrophil extracellular traps (NETs) (NETosis) in the nasopharynx to facilitate bacterial clearance. B cells, stimulated by DCs and T helper cells, differentiate into plasma cells and secrete antibodies. The secreted dimeric IgA binds to the polymeric immunoglobulin receptor and is released into the infected respiratory tissue as secreted IgA.

Figure 3.

Adjuvants with immunological potentials in pertussis vaccines. Numerous adjuvants can potentially be used in pertussis vaccines, including alum, liposome, MF59, CpG ODN, TLR7 agonist, poly (I:C), lipoproteins, AS04, and more.

Table 2.

Preclinical research on novel adjuvants in aP vaccines.

Adjuvant	Animal	Administration	Immune Response	Effect	Potential Application
CpG ODN	Mice	Intraperitoneal	Increase serum anti-PT specific IgG2a and reduce total serum IgE	Inhibit the production of IgE against oral antigens.	Infant priming
CpG ODN	Mice	Intramuscular	Induce Th1/Th2 response and IgG2a/b	Promote the clearance of bacteria in the lower respiratory tract, protect mice against challenge with either PRN or PRN strains of B. pertussis±	Adult boosting
Alum-absorbed TLR7 agonist	Mice	Intramuscular/Intraperitoneal	Induce Th1/Th17 response and IgG2a/b	Increase PT neutralizing antibodies and inhibition of FHA binding to lung epithelium, enhance protective immunity in mouse aerosol challenge model	Infant priming or adult boosting
MF59, Alum+MPLA	Mice	Intramuscular	Induce Th1 response and IgG2a	The neutralizing titer stimulated by Alum + MPLA is higher than that of Alum and MF59, Alum + MPLA and MF59 equally potentiated the inhibitory activity of anti-FHA antibodies	Adult boosting
MPL, LpxL2 LPS	Mice	Subcutaneous	Induce a more Th1 response	Reduce bacterial colonization and type I hypersensitivity, increase serum IL-6 levels	Infant priming or adult boosting
LTK63, LTR72	Mice	Intranasal	LTK63 promote T-cell responses with a mixed Th1–Th2 profile, LTR72 especially at low dose, induce a more polarized Th2 response.	Enhance antigen-specific serum IgG, secretory IgA, and local and systemic T-cell responses	Infant priming or adult boosting
CT	Mice	Intranasal	The IgA responses were significantly reduced, and the IgG responses were not increased	/	No application potential
rCTB	Mice	Intranasal	Develope high levels of anti-PTd serum IgG antibodies, high or moderate levels of anti-FHA serum IgG antibodies and mucosal anti-PTd IgA antibodies in the lungs regardless of rCTB	/	No application potential
CyaA, CyaA*	Mice	Intranasal	Enhance the serum IgG response and the IgA response against Prn in the lungs, but not as effective as LKT63.	/	Infant priming or adult boosting
BLPs	Mice	Intranasal	Induce significant anti-PT IgA and anti-FHA IgA production in nasal irrigation	Prevent bacterial colonization and lung damage	Infant priming or adult boosting
Curdlan	Mice	Intranasal	Not enhance the antibody response to FHA and PT, not affect the ratio of IgG2a to IgG1, induce increase in IL-17A in the lung homogenate.	Promote the clearance of bacteria in respiratory tract	Infant priming or adult boosting
T-vant	Mice	Intranasal	Promoted Th1 and Th17 immune responses and induce mucosal IgA and serum IgG	Expand CD4+T cells, which may be conventional TRM cells, and eliminate B. pertussis in the lungs and nasopharynx	Infant priming
CpG ODN	Mice	Intranasal	Increase serum PT, FHA, and PRN-specific IgG, induce Th1 response	Increase the levels of NO and IFN-γ in macrophages or spleen supernatants, enhance protective immunity in aerosol challenge model	Infant priming or adult boosting
LP1569	Mice	Intraperitoneal	Induce Th1/Th17 response and FHA-specific IgG2a	Confer a higher level of protection against bacterial colonization of the lungs and trachea	Infant priming or adult boosting
LP-GMP	Mice	Intraperitoneal/Intranasal	Intraperitoneal immunity induces Th1/Th17 response and FHA-specific IgG2c, intranasal immunity induces Th17 response	Intraperitoneal immunity promote the clearance of bacteria in the lungs. Intranasal immunity induces IL-17-secreting TRM cells and prevent bacteria from colonizing in the nasal cavity and lungs	Infant priming or adult boosting
2′, 3′-cGAMP, c-di-GMP	Mice	Intranasal	Induce Th1/Th2/Th17 response	Promote the generation of CD4+ TRM populations, inflammatory cell infiltration in the lung and reduce bacterial burden in both the upper and lower respiratory tracts.	Infant priming or adult boosting
Innate defense regulator peptide IDR 1002, a Toll-like receptor-3 agonist poly(I:C), and a polyphosphazene in a fixed combination.	Mice	Intranasal	Induce a more Th1 response and IgA/IgG2a	Produce nasal-secreted sIgA antibody	Infant priming or adult boosting

Aluminum salt adjuvant

Aluminum salt adjuvants were selected for the pertussis vaccine because they have a long history, meet regulatory requirements, and offer logistical advantages in the application of combined vaccines. It is traditionally believed that aluminum salt adjuvants have a depot effect, which can continuously release antigens at the injection site. The combination of aluminum salts and antigens can promote the uptake and presentation by antigen-presenting cells (APCs). Additionally, aluminum adjuvants can also activate the NLRP3 inflammasome and promote the production of IL-1β and IL-18. Aluminum adjuvants promote antibody production and Th2 responses, which are related to humoral immunity.^49,58 Studies have suggested that the type of aluminum adjuvant affects immune responses, with both aluminum hydroxide and aluminum phosphate commonly used to enhance immune responses.^59–62 Among the acellular pertussis vaccines, the three-component vaccine Infanrix^TM or Pediarix^TM, which contains antigens adsorbed on aluminum hydroxide, as well as the five-component Pediacel^TM or Pentacel^TM with antigen adsorbed onto aluminum phosphate, are widely used (Table 1). The effects of DTaP3, which includes the three antigens PT, PRN, and FHA, were compared to those of DTaP5. Morel et al. showed that DTaP3 provided better protection and durability than DTaP5 formulations. Pediacel^TM was less effective than Infanrix^TM at initiating humoral responses, which may be related to the type of adjuvant.⁶³Vaccine antigens are more loosely bound to aluminum phosphate and affect antigen response.⁶³ Denoël et al. also demonstrated that PRN desorbed more easily desorbed from aluminum phosphate adjuvants, influencing the PRN antigen response and mouse lung clearance activity.⁶⁴ This indicates the importance of the formulation process in stimulating an immune response. In the production of adsorbed vaccines, besides the type of aluminum adjuvants, various factors can influence the adsorption of antigens and adjuvants. The pH of the solution determines the surface charge of the adsorbed antigen and adjuvant. When the pH lies between the isoelectric points of the adjuvant and antigen, adsorption can occur via through electrostatic interaction. The presence of ions in the solution, such as phosphates and citrates, can easily lead to the desorption of antigens. For multi-antigen adsorption vaccines like aP vaccines, the sequence of adsorption, such as sequential adsorption, competitive adsorption, and separate adsorption, can also affect the adsorption behavior.⁶⁵

TLR9 agonist

Cytosine-phosphorothioate guanine (CpG) motif-containing oligodeoxynucleotides (ODN) can directly stimulate antigen-presenting cells via TLR9, thereby accelerating the immune response. CpG ODN serves as a potent adjuvant that induces the Th1 response. It promotes the robust generation of cytotoxic T lymphocytes (CTL) and the secretion of IFN-γ, enhancing the specific humoral and cellular immune responses to antigens. CpG ODN 1018 was approved for use in the hepatitis B vaccine HEPLISAV-B, and CpG ODN 2006 was approved for the anthrax vaccine CYFENDUS.^58,66 Sugai et al. included the CpG-ODN in the diphtheria-pertussis-tetanus (DPT) vaccine and evaluated the immunizing effect of the alum and CpG-ODN combination adjuvant. Mice immunized with the DPT vaccine containing CpG-ODN (DPT-alum/ODN) showed a significant reduction in serum levels of total IgE and an increase in serum anti-PT specific IgG2a titers. Additionally, the antibody response to oral ovalbumin (OVA) after vaccination was studied. In the group receiving DPT-alum/ODN, the production of OVA-specific IgE in serum was reduced.⁶⁷ DeJong et al. added CpG 1018 to Tdap, inducing more Th1 antibodies and enhancing the protection of the lower respiratory tract and the bacterial clearance rate. Furthermore, regardless of whether the B. pertussis strain expressed PRN, Tdap + CpG 1018 could provide protection, alleviating concerns that PRN mutants could withstand vaccine-mediated immune responses.⁶⁸ The Phase 1 trial conducted by Dynavax evaluated the safety and immunogenicity of the investigational tetanus/diphtheria/acellular pertussis vaccine combined with the CpG 1018 adjuvant in adults and adolescents, demonstrating acceptable safety and tolerability. The immune response induced by Tdap-1018 3000 μg was similar to or greater than that of Boostrix, which is currently marketed for adults and adolescents.⁶⁹

Although CpG was not used in the pertussis vaccine for special populations, compared with the three-dose administration of the hepatitis B vaccine with aluminum adjuvant, the two-dose administration of hepatitis B vaccine with CPG1018 resulted in a higher serum protection level in the elderly⁷⁰, and no increase in adverse pregnancy outcomes was observed during pregnancy.⁷¹

TLR7 agonist

TLR7 agonists exhibit adjuvant activity and can induce Th1/Th17 responses in various infectious diseases. The combination of small-molecule TLR7 agonists with aluminum adjuvants lessens systemic inflammation and reduces the release of cytokines into the bloodstream, enhancing their safety as adjuvants.⁷²

Misiak et al. added TLR7a (Toll-like receptor 7 agonists) to the aP vaccine containing alum adjuvant, which could transform it from a vaccine that induces a Th2 response to one that induces a Th1/Th17 response for greater protective ability. The alum-TLR7a adjuvanted vaccine was more effective than the alum adjuvanted vaccine and promoted Th1-polarization response with significantly reduced antigen doses. The aluminum-TLR7a adjuvant increased the production of PT-neutralizing antibodies, inhibited the binding of FHA to lung epithelium, and enhanced the protective immunity of the mouse aerosol attack model.^73,74

TLR4 agonist

The TLR4 agonist LPS is a natural component of the outer membrane of B. pertussis and is present in the wP vaccine. It acts as a natural adjuvant and helps induce protective immunity.⁷⁵ 3’-O-deacylated monophosphoryl lipid A (MPL) is a detoxified LPS derivative that can bind to TLR4 and promote the innate immune response. The combination of MPL and aluminum hydroxide adjuvant forms AS04, which has been approved for use in the hepatitis B virus vaccine FENDRIX and the human papillomavirus vaccine CERVARIX.⁵⁸

Geurtsen et al. utilized two LPS analogs: MPL and Neisseria meningitidis LpxL2LPS, to replace aluminum adjuvants in DTaP vaccines. It was found that incorporating an LPS analog into the vaccine reduced lung colonization and induced higher Ptx-specific IgG levels, making the response more characteristic of Th1-type reactions and decreasing type I hypersensitivity reactions.⁷⁶ Agnolon et al. assessed the combination of the emulsion adjuvant MF59 and the TLR4 agonist monophosphoryl lipid A (MPLA) with aluminum hydroxide (alum). The addition of MPLA and MF59 increased the IgG_2a antibody titer, stimulated the IgG_2a/Th1 response, enhanced the neutralizing activity of DT, and blocked the binding of FHA to human lung epithelial cells.⁷⁷ Mitchell et al. believed that the TLR4 stimulating function of B. pertussis is one of the properties lost in the transformation of wP into aP vaccines with minimal endotoxin, and TLR4 is essential for immunity. MPL has the adjuvant characteristics that may be necessary for modified pertussis vaccines, including inhibiting reactivity, providing longer-lasting immunity, and reducing Th2 bias. Moreover, AS04 has a good safety profile, and vaccination with the AS04-HPV-16/18 vaccine within 60 d before conception until delivery does not increase the risk of teratogenicity.⁷⁸ The AS04 adjuvant is highly suitable for use in the application of pertussis vaccines.⁷⁹

TLR2 agonist

B. pertussis expresses many lipoproteins, which act as TLR2 agonists and also possess antigenic properties.⁷² Dunne et al. purified a novel TLR2-activated lipoprotein BP1569 from B. pertussis, demonstrating that the corresponding synthetic lipopeptide LP1569 effectively stimulated the immune system and exhibited adjuvant properties, inducing Th1 and Th17 responses. It proved to be more effective than alum-based vaccines in preventing colonization of B. pertussis in mice.⁸⁰

MF59

MF59 consists of squalene, polysorbate 80, sorbitol trioleate, and trisodium dehydrated citrate. It has been included in the licensed products across 30 countries and is primarily used for influenza vaccines. MF59 promotes the absorption of antigens and the recruitment of immune cells at the injection site, enhances the transportation of antigens to the drainage lymph nodes, and strengthens the immune responses of Th1 and Th2.⁵⁸

Agnolon et al. found that the combination of MF59 and MPLA with Alum was similar, each capable of increasing the titer of IgG2a antibody, stimulating the Th1 response, and blocking the binding of FHA to human lung epithelial cells by serum antibodies. In comparison to the combination of MPLA and Alum, MF59 proved to be the most effective adjuvant for TT and PRN antigens.⁷⁷

MF59 has been used in the Flaud® influenza vaccine for people over 65 y old and shows great potential to induce strong immune response in the elderly. Studies on pregnant women who received Focetria® (MF59 adjuvant A/H1N1 pandemic influenza vaccine) demonstrated that the MF59 adjuvant A/H1N1 pandemic influenza vaccine was safe during pregnancy.⁸¹ This suggests that MF59 may also play a role in booster vaccination for the pertussis vaccine in adults.

Mucosal adjuvants for aP vaccines

Pertussis affects the upper respiratory tract. Thus, respiratory mucosal immunization is critical in the prevention of pertussis infection.⁸² Human pertussis infection produces IgA in nasal secretions. Although there is no evidence that IgA deficiency is linked to pertussis infection, anti-pertussis secretory IgA (sIgA) in nasal lotions and serum IgA from convalescent patients have been shown to inhibit the adhesion of pertussis bacteria to human respiratory epithelial cells.^83,84 Aside from IgA, tissue-resident memory T (T_RM) cells play a critical role in mucosal immunity. In a mouse model, IL-17-secreting CD4⁺ T_RM cells migrate to the nasal tissues during the initial infection and subsequently mediate the recruitment of Siglec-F⁺ neutrophils. Additionally, the local expansion of IL-17-CD4⁺ T_RM cells during re-infection helped clear B. pertussis from the nasal cavity (Figure 2).^85–87 The intramuscularly administered aP vaccine with aluminum adjuvant induces Th2 immunity but does not promote sIgA or T_RM cells in the respiratory tract.⁸⁴ Therefore, mucosal immunity may be more effective in preventing nasal colonization and infection. The development of new adjuvants aims to facilitate intranasal immunization to prevent bacterial colonization in the nasal cavity and lung infections.⁵²

Cholera toxin (CT), E. coli heat-unstable enterotoxin (LT), and their gene-detoxifying mutants are commonly used as mucosal adjuvants. Ryan et al. combined an aP vaccine with the nontoxic mutant LKT63 or the partially enzymatically active LTR72 mutant for intranasal immunization. They found that both induced enhancements in antigen-specific serum IgG and secreted IgA, along with accelerating the clearance of lung bacteria. The nontoxic mutant LTK63 boosted both Th1 and Th2 responses, while the partially enzyme-active LTR72 mutant favored Th2 responses.^83,88 Although LT can enhance the immune response of the intranasal aP vaccine, the immune effect of CT as an adjuvant for the pertussis vaccine is less satisfactory. Herland Berstad et al. found that the combined intranasal immunization with CT and B. pertussis failed to enhance the IgA response and significantly reduced it, likely due to the interaction of CT with the nasal epithelium, competing for components of the B. pertussis preparation.⁸⁹ Isaka et al. also found that when recombinant cholera toxin B subunit (rCTB) was used in combination with the DPT vaccine for intranasal immunization, mucosal adjuvants for pertussis toxoid (PTd) and formalin-treated filamentous hemagglutinin (fFHA) showed no significant effect.⁹⁰ Orr et al. investigated the effects of immunization when administering adenylate cyclase toxin (CyaA) from B. pertussis intranasally to mice as a mucosal adjuvant. The co-administration of enzymatically active CyaA and CyaA lacking adenylate cyclase activity (CyaA*) with Prn significantly enhanced serum IgG responses. Immunization with Prn and CyaA* resulted in a notably improved lung anti-Prn IgA response compared to Prn and CyaA. However, neither adjuvant was as effective as LKT63.⁹¹ Although the adjuvanticity of CT and LT is widely studied and recognized, they may cause Bell’s palsy, which limits their use in human vaccines.^83,92 Safer and more effective mucosal adjuvants are being developed. Shi et al. introduced a novel bacterial-like particle (BLP) mucosal adjuvant based on the food-grade bacterium Lactococcus lactis to pertussis antigens, such as PT, FHA, and PRN, and conducted intraperitoneal and intranasal immunization. They found that compared with alum, intranasal immunization with BLPs resulted in significant anti-PT IgA and anti-FHA IgA production in nasal irrigation and was more effective at preventing bacterial colonization and lung damage.⁹³ Boehm et al. formulated DTaP vaccines that contain the curdlan adjuvant, a 1,3 β-glucan derived from Alcaligenes faecalis. The curdlan adjuvant facilitated the localization of DTaP particles to the upper respiratory tract. Although the curdlan adjuvant did not boost the antibody response against FHA and PT compared to the original DTaP vaccine, the intranasally immunized vaccine formulated with curdlan adjuvant induced a significant increase in IL-17A in the lung homogenate. Whether administered intraperitoneally or intranasally with the curdlan adjuvant formulated vaccine, no significant expansion of T_RM cells was seen in the lungs. The intranasally administered DTaP vaccine combined with curdlan adjuvant also cleared B. pertussis from the respiratory tract.⁹⁴ Galeas-Pena et al. developed an intranasal vaccine composed of aP antigens and T-vant, a novel adjuvant derived from bacterial outer membrane vesicles. This vaccine promoted Th1 and Th17 immune responses, expanded CD4⁺T cells, which may be conventional T_RM cells, and eliminated B. pertussis in the lungs and nasopharynx when delivered intranasally. In addition, it could also induce mucosal IgA and serum IgG.⁹⁵

Liposome adjuvants, STING and TLR agonists, and their combinations can also serve as mucosal adjuvants. Asokanathan et al. demonstrated that when aluminum hydroxide is combined with CpG-ODN, the pertussis vaccine can increase serum PT, FHA, and PRN-specific IgG antibodies. The levels of NO and IFN-γ in macrophages or spleen supernatants were significantly increased, indicating that the immune response shifted to Th1.⁹⁶ Based on the research on synthetic lipopeptide LP1569, Allen et al. then continued to use a new adjuvant, LP-GMP, which contains LP1569 and c-di-GMP. C-di-GMP acts as an intracellular interferon gene receptor stimulator (STING). LP-GMP has the ability to activate DCs in both mice and humans, promoting effective Th1 and Th17 immunity. When co-immunized with pertussis antigens, LP-GMP can prevent nasal colonization and lung infections caused by B. pertussis, and it induces more respiratory T_RM cells than intraperitoneal immunity during intranasal vaccination, a large proportion of which are lung tissue-resident CD4⁺ cells secreting IL-17.^85,87 Jiang et al. found that nasal administration of the agonist c-di-GMP as an adjuvant for the aP vaccine induced Th1 and Th17 immunity and significantly increased CD4⁺ T_RM cells in the lungs. The aP vaccine group with the c-di-GMP adjuvant demonstrated better protection than the other groups, including the aP vaccine formulated with aluminum hydroxide. Furthermore, the infiltration of inflammatory cells in the lungs was reduced, as was the bacterial load in the respiratory tracts.⁹⁷ Aibna et al. combined PRN, FHA 2/3, and pertussis toxin mutants with a triple adjuvant, which includes the Toll-like receptor (TLR)-3 agonist poly (I:C), a host innate defense peptide (IDR-1002), a polyphosphazene carrier system (PCEP), and cationic DDAB/DOPE liposomes for intranasal immunity. This vaccine promotes the production of Th1-type immunity with high IgG2a and IgA serum antibody titers and produces nasal-secreted sIgA antibody after a single dose.⁹⁸

The stability of the pertussis combination vaccine

As seen above, the combined pertussis vaccine typically includes diphtheria, tetanus toxoid, purified subunit protein from pertussis bacteria, inactivated poliovirus antigen, and others. This combination vaccine often requires multiple injections and is formulated as a liquid preparation, which imposes high standards for the stability of the vaccine during long-term storage.⁹⁹ The stability of the combination vaccine is quite complex and relies on the interactions among its various components. When the vaccine includes adjuvants, the pertussis antigen, diphtheria toxoid, and hepatitis B antigen compete for binding sites on the adjuvant, leading to situations where other antigens often displace diphtheria toxoid and result in reduced efficacy of diphtheria toxoid.¹⁰⁰ Kalbfleisch et al. demonstrated that nearly 90% of the monovalent antigens DT, TT, and FHA were adsorbed onto aluminum phosphate, while PRN, PT, and FIM exhibited lower adsorption rates. Following adsorption with aluminum phosphate, DT and TT displayed more noticeable structural changes than the other antigens.¹⁰¹ Duprez et al. characterized the acellular pertussis vaccine Tdap, which contains genetically modified pertussis toxin (gdPT) and TLR agonists (E6020 and CpG) adsorbed to the AlOOH adjuvant. In comparison with Tdap-AlOOH and Tdap-E6020 (TLR4 agonist)-AlOOH, Tdap-CPG-AlOOH exhibited better thermal stability, and FTIR analyses indicated that the secondary structure of gdPT remained essentially unchanged after being adsorbed by AlOOH, compared to PT.¹⁰² The desorption kinetics of the antigen on the aluminum adjuvant will influence the storage stability. When the Al(OH)₃-based Infanrix vaccine was stored at 4°C for up to 3 y, all batches indicated that the desorption rates of PT, FHA, and PRN were lower than 3%, 0.8%, and 0.1%, respectively. When the antigen is adsorbed on AlPO₄, the maximum loss of PT is 11–12% after 18–24 months, while PRN gradually desorbs to a level of 60% after 2 y. Adsorption to aluminum phosphate may lead to poor protection against intranasal pertussis challenge in mice.⁶⁴

Additionally, the presence of preservatives and extreme temperatures can impact the stability of the pertussis combined vaccine. Both diphtheria and tetanus toxoid tend to remain relatively stable at higher temperatures, while vaccine efficacy is more likely to diminish under frozen conditions. Both elevated and freezing temperatures affect the pertussis vaccine, but the effects of freezing are more pronounced.¹⁰⁰ Pertussis ingredients are stored at 22–25°C for 2–8 weeks and still maintain 80% of their initial efficacy. When the vaccine is kept at −20°C for 15 d, the effectiveness of pertussis ingredients reduces by about 50%.¹⁰⁰ Studies indicate that freeze injury of vaccines is associated with aluminum salt adjuvants. The presence of aluminum salt adjuvant increases the vaccine’s sensitivity to freezing temperatures. Freezing the aluminum salts destroys the vaccine, while freeze-induced damage to the hydroxyl groups on the surface of the aluminum adjuvant leads to reduced repulsion between aluminum particles, facilitating the formation of aggregates. The outcome is diminished vaccine effectiveness.^103,104 Xue et al. found that adding propylene glycol excipients to DTaP and DTwP vaccines can protect the vaccine from freezing damage without affecting the pH, particle size distribution, and efficacy of the vaccine.¹⁰⁵

Overall, in the combined pertussis vaccine, the components of tetanus and diphtheria exhibit greater stability than those of pertussis, and the vaccine’s overall stability is influenced by various factors such as temperature, excipients, adjuvants, and others. Due to the complexity of the combined vaccine, no definitive conclusions have been reached yet.¹⁰⁰

Conclusions and perspectives

This article provides a comprehensive overview of current combination vaccines containing pertussis antigens. It also explores approaches to enhance the immunogenicity of aP vaccines by incorporating adjuvants that promote Th1 and Th17 responses. We discuss examples of promising adjuvants in pertussis combination vaccines, such as CpG-ODN, MPL, and LPS, offering insights for selecting new adjuvants in aP vaccine formulations. Furthermore, transitioning from intramuscular to nasally administered pertussis vaccines could further improve efficacy. This paper summarizes the application of various mucosal adjuvants in pertussis vaccines, highlighting their potential to enhance immune responses.

The ideal adjuvant should possess extensive safety, be easily manufactured and administered, effectively activate both humoral and cellular immune responses, and minimize adverse reactions. This article outlines various new aP vaccine adjuvants demonstrating protective effects in preclinical animal trials. Among them, CpG, TLR7a, LP1569, and MPL can prevent B. pertussis from colonizing in the lungs or trachea when administered intramuscularly or intraperitoneally, while mucosal adjuvants administered intranasally such as LP-GMP and c-di-GMP can even induce mucosal immunity in the nasal cavity and reduce bacterial load. The comparison between MF59 and aluminum hydroxide +MPLA in the TdaP vaccine indicated that the immune response induced by aluminum hydroxide +MPLA took effect more quickly.⁷⁷Additionally, the new adjuvants were not directly compared in the pertussis vaccine. However, previous studies show that most TLR agonists can induce antigen-specific CD4 T cells, but only stronger TLR7/8 and TLR3 agonists can induce CD8 cytotoxic T cells.¹⁰⁶ In addition to the immune effects, the complexity of adjuvant production, stability, and the potential for inducing immune tolerance all require further evaluation to prevent clinical limitations. TLR agonists tested in clinical trials and marketed vaccines exhibit stable chemical properties and low production costs, making them promising candidates for development. Unlike traditional aluminum adjuvants, TLR activators do not need to be adsorbed with antigens, which appears to reduce development complexity. However, research on the stability and immunogenicity resulting from antigen-adjuvant interactions remains essential. The combined use of multiple adjuvants offers another promising avenue for future aP vaccine development. We believe that the use TLR agonists, especially those with proven safety like CpG and AS04, in pertussis vaccines will become a common trend. Notably, while the new adjuvant helps induce immune enhancement and long-term protection, it remains unclear whether it can overcome the selective pressure from antigen-deficient strains. More study is necessary in this aspect.

The discovery and selection of new adjuvants have traditionally depended on empirical screening and optimization of candidate compounds, with a limited understanding of their immune mechanisms.¹⁰⁷ Moreover, the interaction of new adjuvants with other vaccine components must be carefully considered.^10,52,108 These conventional approaches are often expensive, time-consuming, and difficult to align with vaccine safety and efficacy standards without a clear understanding of adjuvant mechanisms and toxicity. As a result, developing aP combination vaccines with new adjuvants remains a complex and protracted process. Recent advancements in artificial intelligence (AI) have opened new avenues for adjuvant discovery and selection. AI utilizes virtual assess to evaluate the biological activity of candidate compounds based on their chemical structures and predict their interactions with specific targets, allowing for the rapid identification of active adjuvants.^109,110 Furthermore, AI can examine the relationship between the physicochemical properties and biological activity of adjuvants through quantitative structure-activity relationship (QSAR) models, facilitating the rational optimization of adjuvant formulations.^109,110 Although AI technology has not yet been extensively applied to developing new adjuvants for aP vaccines, its potential to transform this field is undeniable. The integration of AI is set to drive significant advancements in the design and optimization of aP vaccines.

Moreover, efforts must be made to increase pertussis vaccine coverage in developing countries and reduce infant mortality. Maintaining the stability of pertussis vaccines in remote areas with limited resources requires substantial investment in cold chain infrastructure. Advancements in vaccine stability through advancements in excipients, adjuvants, and formulation technologies can improve vaccine stability and help ensure that resource-constrained developing countries gain better access to effective vaccines.

The ideal pertussis vaccine should demonstrate excellent safety and stability, elicit both humoral and cellular immunity, provide long-term protection, and not interfere with the effectiveness of other vaccine components. A deeper understanding of the components, mechanisms, and types of adjuvants used in pertussis combination vaccines can accelerate the development and clinical application of improved pertussis vaccines. By addressing these challenges, we can move closer to achieving a highly effective and globally accessible pertussis vaccine.

Biographies

Ge Yu obtained her B.S. in Chemical Technology and Ph.D. in Chemical Engineering from Dalian University of Technology (DUT). In 2024, she joined Changchun BCHT Biotechnology Co., Ltd. Her research interests focus on vaccine formulation, vaccine adjuvants, and nano-bio interaction.

Changying Xue obtained her Ph.D. in Chemical and Biomolecular Engineering from the National University of Singapore (NUS), and both a B.S. and an M.S. in Chemical Engineering from Dalian University of Technology (DUT). She had her postdoctoral training at the University of Illinois at Urbana-Champaign (UIUC). In 2016, she began her academic career as a professor at the School of Bioengineering at DUT. Her research interests focus on biomaterials, vaccine adjuvants, and surface chemistry.

Bingbing Sun obtained both a B.S. and an M.S. in Chemical Engineering from Dalian University of Technology (DUT), and a Ph.D. in Chemical Engineering from the University of Washington. He completed his postdoctoral training at the University of California, Los Angeles (UCLA). In 2016, he began his academic career at the School of Chemical Engineering at DUT, where he currently holds the position of full professor. His research interests include vaccine adjuvants, biomaterials, nano-bio interfaces, and immunoengineering.

Funding Statement

This work was supported by the National Key Research and Development Program of China [2022YFC2304305] and the National Natural Science Foundation of China [U22A20455].

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The data summarized in this review are from published articles and are publicly available.

Ethical approval

This work does not involve human participants, and it was not necessary an ethical approval by the ethic committee.