Applying lessons from human papillomavirus vaccines to the development of vaccines against Chlamydia trachomatis

Abstract

Introduction:

Chlamydia trachomatis (Ct), the most common bacterial STI, leads to pelvic inflammatory disease, infertility, and ectopic pregnancy in women. In this Perspective, we discuss the successful human papillomavirus (HPV) vaccine as a case-study to inform Ct vaccine efforts.

Areas covered:

The immunological basis of HPV vaccine-elicited protection is high-titer, long-lasting antibody responses in the genital tract which provides sterilizing immunity. These antibodies are elicited through parenteral administration of a subunit vaccine based on virus-like particles (VLPs) of HPV. We present three lessons learned from the successful HPV vaccine efforts: (1) antibodies alone can be sufficient to provide protection from STIs in the genital tract, (2) the successful generation of high antibody levels is due to the multivalent structure of HPV VLPs, (3) major challenges exist in designing vaccines that elicit appropriate effector T cells in the genital tract. We then discuss the possibility of antibody-based immunity for Ct.

Expert Opinion/Commentary:

In this Perspective, we present a case for developing antibody-eliciting vaccines, similar to the HPV vaccine, for Ct. Basic research into the mechanisms of Ct entry into host cells will reveal new vaccine targets, which may be antigens against which antibodies are not normally elicited during natural infection.

1.0. INTRODUCTION

The estimated global prevalence of Chlamydia trachomatis (Ct) among women and men 15–49 years old is 4.2% and 2.7%, respectively, making it the most common bacterial sexually transmitted infection [1]. Ct has been identified by the World Health Organization (WHO) and the U.S. National Institutes of Health (NIH) as a STI for which a vaccine is needed. One vaccine candidate has entered Phase I clinical trials [2] and others are in the pipeline, although historical experience of STI vaccine trials in general, and Ct vaccines specifically [3], urge cautious optimism. The experience developing the human papillomavirus (HPV) vaccine provides important lessons for protecting against STIs. The prophylactic HPV vaccine has been remarkably successful; it elicits potent and long-lasting protection against HPV infection and markers of cervical disease (e.g. CIN 2, 3). In this Perspective, we review the immunological basis for the success of the HPV vaccine and discuss how this knowledge can be applied to Ct vaccine efforts.

2.0. HPV vaccines.

HPV is the most common sexually transmitted infection despite a decrease in prevalence of infection after the introduction of the HPV vaccine [4]. In the United States alone, over 6 million new HPV infections are reported each year and greater than 75 million people are estimated to be currently infected [5]. HPV infection can cause a variety of different pathologies, but the most concerning is HPV-associated cervical cancer. Persistent infection with one of the high-risk HPV types (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68) can cause cervical and other mucosal cancers. These high-risk HPV types account for 5.2% of all cancers world-wide [6]. In the early 1990s, it was discovered that the HPV major capsid protein, L1, could self-assemble into virus-like particles (VLPs) [7]. VLPs structurally resemble authentic HPV virions, but are not infectious because they lack a viral genome. In subsequent studies, it was shown that parenteral administration of HPV VLPs could elicit high-titer HPV-neutralizing antibodies [8]. Clinical trials demonstrated that HPV VLP-based vaccines could effectively prevent HPV infection and disease caused by the HPV types targeted by the vaccines [9]. These studies paved the way for clinical approval of the HPV vaccines Gardasil (Merck) and Cervarix (GSK). Both Gardasil and Cervarix include VLPs derived from the L1 protein of high risk HPV types 16 and 18. Gardasil additionally includes HPV 6 and 11, low risk HPV types that cause genital warts. A next-generation HPV vaccine, Gardasil-9, was released by Merck in 2014 and includes five additional high-risk HPV types (31, 33, 45, 52, and 58).

2.1. The basis for protection mediated by HPV vaccines.

There is strong evidence that the protection provided by the VLP-based HPV vaccines is mediated by neutralizing antibodies. Immunization with HPV L1 VLPs elicits high-titer antibody responses [7], and passive transfer of immune sera (from animals or humans) can provide sterilizing protection from cervicovaginal or mucosal HPV challenge in animal models [10, 11, 12]. Vaccine-induced HPV-specific IgG and IgA responses are not only present in the serum, but are also detected in the genital tract [13]. The source of antibodies in cervico-vaginal secretions is both local antibody-producing plasma cells and antibodies that cross into the mucosal barrier from the plasma by active transcytosis or exudation at breaches in the cervicovaginal epithelium [14]. Immune sera from rabbits vaccinated with HPV VLP vaccines blocks HPV infection at two steps: binding of HPV virions to the basement membrane, and binding of HPV to the surface of epithelial cells [15]. Interestingly, in one study 10–30% of vaccinated women do not have detectable levels of antibody to HPV in cervicovaginal secretions [13]. However, in one study, mice received antibodies via intraperitoneal transfer of immune sera obtained from Gardasil immunized mice, and the amount of sera needed to see protection against genital tract challenge with HPV was less than 1/1000^th of a uL of sera [10]. This, along with no evidence of breakthrough HPV infection in vaccinated individuals, suggests that the minimum amount of antibody necessary for protection against HPV infection is exceedingly small. It remains to be seen whether such a small effective dose of antibodies is sufficient to protect against other STIs.

It is important to note that, although the HPV L1 VLPs can elicit T cell responses against L1 (the only protein component of the VLPs), the preponderance of evidence suggests that T cells are not responsible for the protection elicited by the vaccine [16]. This is due to the unique HPV replication cycle, whereby HPV gains access to basal epithelial cells (the target host cell) through microabrasions, initiate cell cycle progression through the production of viral proteins E6 and E7, and only after terminal differentiation of the infected epithelial cells produces L1 and other structural proteins that assemble into virions [17]. Thus, any T cells elicited by the vaccine would be specific for the HPV L1 protein, which is not expressed in the basal epithelial cells. Basal epithelial cells harbor persistent HPV after infection, and they are the origin of neoplastic lesions. Indeed, clinical studies of HPV vaccinated individuals showed that vaccination fails to induce regression of previously infected lesions [18], indicating that the T cells that are elicited in response to vaccination with HPV VLPs are not able to act on infected cells to provide protection against neoplastic lesions.

2.2. Challenges in developing therapeutic HPV vaccines

While the success in developing prophylactic HPV VLP-based vaccines (i.e. those vaccines that prevent HPV infection) encourages efforts to develop vaccines for other STIs, the challenges in developing therapeutic HPV vaccines (i.e. those that would treat HPV-infected lesions or cervical neoplasia) are also instructive. As mentioned above, the current HPV VLP-based vaccines are prophylactic and do not exhibit any value for therapeutic treatment of current HPV infections or cervical cancer. Attempts to develop therapeutic HPV vaccines have focused on generating cell-mediated immune responses against the HPV proteins that are expressed in the basal epithelium, particularly the viral oncoproteins E6 and E7 from high-risk HPV types. Various vaccine strategies to target E6 and/or E7 have been employed in clinical settings, including peptides [19], fusion proteins [20], vector-based strategies [21, 22], and DNA vaccines [23, 24]. There have been modest successes in phase 2 clinical studies, including a DNA vaccine targeting E6 and E7 proteins that showed efficacy against HPV-16 and HPV-18 associated CIN2/3, but was not as effective as surgical resection [24]. No therapeutic HPV vaccine has yet been evaluated in a randomized phase 3 trial.

Why have therapeutic HPV vaccines shown limited efficacy? One possibility is that most HPV therapeutic vaccines have been administered systemically or at remote mucosal sites. It is likely that vaccines that can induce local cellular immune responses in the genital tract will be advantageous for targeting HPV lesions. While the best route for eliciting strong T cell responses in the genital tract is still debated, local genital immunization [25] or topical application of an adjuvant [20] or chemokines [26] may promote local proliferation or recruitment of T cells. Even when local T cell responses are successfully induced, the breadth of T cell responses is also likely to be an important factor in determining efficacy. For example, for therapeutic HPV vaccines, inadvertent concomitant induction of regulatory T cells has been associated with treatment failure—i.e. no regression of neoplastic lesions [20, 27]. Thus, experience with therapeutic HPV vaccines has illustrated the challenges in eliciting the appropriate protective T cell subsets to the specific location of the genital tract.

2.3. Lessons learned from HPV vaccines.

The successful production of prophylactic HPV vaccines and the challenges in developing therapeutic vaccines against HPV illustrate several lessons that can be applied generally to vaccination efforts for other sexually transmitted pathogens. First, and most importantly, the HPV vaccine has demonstrated that antibodies alone are sufficient to protect against an STI. There are several specific features of the mechanism of HPV infection that may make this virus particularly susceptible to antibody-mediated neutralization (reviewed by [28]), but, nevertheless, the HPV vaccine is capable of eliciting high enough levels of antibody in the genital tract to provide sterilizing immunity. Importantly, these levels of antibody in the genital tract can be achieved by parenteral administration rather than by using a specialized approach to elicit mucosal antibodies. Second, the successful generation of high levels of antibodies against HPV is attributed to the multivalent (a word which here means “repetitive” or “having multiple sites”) structure of HPV VLPs [16]. Third, there are major challenges in designing successful vaccines for inducing effector T cell responses against STI antigens in the genital tract. Therapeutic vaccines for HPV have appropriately targeted the viral oncoproteins. However, even when the target antigen is known, there are no straightforward techniques to direct effector T cell responses to the genital tract while, at the same time, avoiding activation of regulatory T cell subsets.

3.0. Vaccines for Chlamydia trachomatis.

Rather than exhaustively reviewing all historical and current Ct vaccine efforts, we refer the reader to three excellent recent reviews that provide a detailed discussion of Ct vaccine efforts [3, 29, 30]. Additionally, we refer the reader to a recent report on Ct vaccine strategies. This report derives from a workshop that was convened in May 2015 at the National Institute of Allergy and Infectious Diseases (NIAID) to provide an assessment of current vaccine efforts and develop recommendations for future Ct vaccine efforts [2]. The report provides recommendations on appropriate animal models, areas of future research that need to be investigated, and desired features of future Ct vaccines. The workshop concluded that a successful Ct vaccine would likely need to elicit both Ct-specific CD4+ T cell responses in the genital tract and strong antibody responses. This consensus is based on current evidence in animal models of infections, which show that CD4+ T cells are necessary for clearing primary infection and that antibody can protect against subsequent infection [2]. The workshop participants also questioned the ability of systemic immunization to be an effective mode of delivery for a vaccine capable of conferring protection in the genital tract, a viewpoint expressed previously [3]. Many of these concerns and recommendations mirror those shared by the HPV research community prior to the demonstrated success of the antibody-based HPV vaccine [31]. Given the profound success of the HPV VLP-based vaccines, we posit that a parenteral vaccine that elicits high-titer antibody may also be sufficient to induce sterilizing immunity against genital infection with Ct.

3.1. Can a vaccine that mediates protection through antibody effectively prevent primary Ct infection?

In order to consider the possibility of whether vaccine-induced antibody might be sufficient to protect against Ct, we must first consider the life-cycle of Ct in host cells in the female genital tract. Although Ct is a bacterium, it shares many features with HPV: namely its mode of transmission, biological niche, ability to cause long-term sequelae in the female reproductive tract, and primary site of initial host cell infection in squamocolumnar epithelial cells in the transition zone of the cervix [32] (Table 1). However, Ct is more antigenically complex than HPV. Ct exists primarily in two forms: infectious elementary bodies (EBs) and metabolically active reticulate bodies (RBs). The infectious life-cycle begins when EBs attach to the surface of host cells and gain entry. Once EBs enter into the host cell, they establish a cytoplasmic inclusion by actively preventing the fusion of the endosome with the lysosome. In the inclusion, the Ct EBs differentiate into the metabolically active RB form and replicate. RBs differentiate back into EBs prior to release from the cell through either lysis or extrusion of the inclusion vacuole [33].

Table 1.

Features of HPV and Ct relevant to vaccine design

Feature	HPV	Chlamydia trachomatis
Transmission route	Sexual	Sexual
Site of initial infection in female genital tract1	Squamocolumnar epithelial cells in the transition zone of the cervix	Cervical epithelial cells
Disease sequelae	Cervical intraepithelial neoplasia, cervical cancer,	Pelvic Inflammatory Disease, ectopic pregnancy, infertility
Size of proteome	8 proteins (2 structural)	~903 predicted proteins (17 predicted EB surface expressed)
Pre-clinical animal models	Mice, rabbits, non-human primates	Mice, minipigs, non-human primates
Mechanism of protection after natural infection	Unknown	IFNγ producing CD4+ T cells in humans and mouse models
Strength of evidence for antibody-mediated protection after natural infection in humans	Weak	Weak
Strength of evidence for antibody-mediated protection after vaccination in humans	Strong	No human vaccine yet available

¹The site of initial infection in the female genital tract is thought to be the squamocolumnar epithelial cells in the transition zone of the cervix for both HPV and Ct. For HPV, this is supported by the origin of precancers and cervical intraepithelial neoplasias near the ectoendocervical squamocolumnar junction of the cervix. However, for Ct we have not seen definitive evidence of the particular cell type that is a site for initial infection.

The antibody-mediated protection provided by the HPV VLP-based vaccines is likely mediated by direct neutralization of the viral particles, which prevent adhesion and entry of the virions into the basal epithelial cells. In the case of Ct, antibodies could mediate protection by (1) neutralization of the EBs to prevent adhesion and/or entry and therefore prevent obligate intracellular bacterial replication and/or (2) opsonization of extracellular infectious EBs and increased opsonophagocytic clearance of Ct. We will consider each of these mechanisms in turn.

3.2. Antibody-mediated prevention of Ct EB entry into host cells.

Similar to the activity of HPV-neutralizing antibodies, antibodies that bind to and/or inactivate the function of molecules necessary for adhesion and/or entry of Ct EBs into the host cell could protect against Ct infection. Whereas HPV virions consist of two structural proteins (each of which can be targeted by neutralizing antibodies), Ct EBs, by one account, express 17 different proteins on their surface, and many of these proteins are extremely large [34]. Thus, unlike HPV, it is not entirely obvious which Ct antigens could serve as the basis for a vaccine that elicits blocking antibodies. One effective tool for vetting possible vaccine antigens is by testing monoclonal antibodies or sera for neutralizing activity by cell culture-based Ct neutralization assays, and then using the targets of those antibodies as vaccine antigens. This line of experimentation has identified the major outer membrane protein (MOMP), the polymorphic membrane proteins (Pmp), and others (PorB, OmcB, etc.) as possible vaccine antigens [3] [35, 36, 37] [38] [39, 40]. While many of these vaccine candidates elicit antibodies that show potent neutralizing activity in cell culture, to date no antibody-based vaccine has demonstrated protection in animal challenge models. There are several possible explanations for this observation: (1) antibody levels elicited by the immunization strategies employed so far are not of sufficient titer in the genital tract to provide measurable protection, (2) the antibodies generated are not the correct isotype to mediate protection, (3) the antibodies do not target the correct epitopes, (4) the animal model or route of infection being used is not appropriate for assessing protection, and/or (5) antibodies that are sufficient to block entry in cell culture are not sufficient in the more complex in vivo environment. The relative role of each of these possibilities remains to be determined by future studies. Moreover, ongoing research into the mechanisms of host cell entry for Ct may reveal new and clear targets for neutralizing antibody-generating vaccines.

3.3. Opsonization and increased clearance of Ct EBs by phagocytes.

Antibody could also prevent Ct infection by serving as an opsonin, thereby enhancing clearance of extracellular bacteria by phagocytes. A recent report indicates that IFN-γ stimulated neutrophils can kill Ct in vitro, and this killing requires Ct-specific serum [41]. This suggests that vaccine-induced antibody against Ct might also increase opsonophagocytic killing of Ct by neutrophils and macrophages. Conversely, there are also reports that Ct can infect cultured macrophages in vitro [42, 43, 44]. Whether or not these phenotypes hold true in vivo remains to be determined. It will be important to fully investigate the activity of any antibodies elicited by vaccine candidates for their opsonization versus enhanced infection capabilities in vitro and in vivo.

4.0. Selecting protective antigens for an antibody-eliciting Ct vaccine.

Most Ct vaccine efforts have focused on eliciting antibody responses to naturally antigenic proteins. However, since natural infection with Ct does not confer lifelong immunity, it may be best to consider antigens that are not immunogenic during natural infection. As an example, recent efforts to generate a next generation HPV vaccine have focused on eliciting strong antibody responses to the minor HPV structural protein L2 [45]. L2 is not immunogenic during natural HPV infection, and yet antibodies to L2 are neutralizing and protective [46, 47]. L2 is considered a “cryptic epitope”, an epitope that is normally not immunogenic because it is exposed only transiently during entry into the host cell. However, if antibodies can be generated to this epitope (i.e. by displaying the L2 epitope on an immunogenic vaccine platform), then those antibodies are neutralizing and are sufficient to protect against infection [48]. Similarly, it is possible that the best antigen to target for a Ct vaccine that relies on an antibody-based mechanism of protection will be a similar cryptic epitope. Features to look for in a cryptic epitope are (1) the antigen or epitope is highly conserved among different types of Ct, (2) it is not normally immunogenic during natural infection, and (3) it is involved in an essential step in the bacterial infection process such as entry into host cells. Some possible Ct proteins that may have cryptic epitopes are MOMP, PorB, Pmps, and the proteins of the T3SS apparatus.

It is notable that some of the Ct EB surface-exposed proteins serve redundant functions. This provides a selective advantage to the pathogen, allowing it to escape antibody-mediated neutralization, but makes identifying appropriate neutralizing targets for vaccine design difficult. The Pmp proteins are a clear example, with these proteins serving redundant adhesion functions in experiments carried out in cell culture [49]. Because of this redundancy, an antibody-based approach for vaccination against Ct may require the inclusion of multiple targets. Some protein epitopes may also serve as “immunological decoys” [50], displaying highly variable, immunogenic domains that drive the antibody response toward non-essential regions of the protein while avoiding antibody responses that may prevent infection. Selecting protective antigens for Ct will therefore require additional research into the mechanisms of adhesion and entry.

5.0. Advantages of antibody-eliciting vaccines for Ct

There are significant advantages of antibody-eliciting vaccines, including: (1) the possibility of single-dose vaccines, (2) multi-component vaccine formulations that target multiple antigens and serovars, (3) fine-targeting of short peptide epitopes to avoid “immunological decoy” problems or target normally non-immunogenic epitopes, (4) the possibility of a simplified pre-clinical pipeline.

Antibody-eliciting vaccines have the potential to be single-dose vaccines. Although HPV vaccines are currently administered in two or three doses, evidence is emerging that even a single dose of HPV vaccine is sufficient to provide long-lasting protection [51, 52, 53, 54]. Indeed, a randomized controlled trial of one-dose HPV vaccine is currently underway (ClinicalTrials.gov: #NCT03180034). This potent immunogenicity is unprecedented for subunit vaccines, but evidence is emerging that this strong immunogenicity is a common feature of vaccines that present antigen on a multivalent platform, such as VLPs. For example, mice immunized with a bacteriophage VLP displaying the HPV L2 epitope continued to have high titer antibodies to the displayed peptide for the life-time of the mouse [55]. This feature of antibody-eliciting vaccines is especially important when considering Ct vaccines that will be administered in resource-poor areas of the world, where administering multi-dose vaccines is logistically challenging.

Vaccines that mediate protection only through antibody lend themselves readily to multicomponent vaccine formulations. Gardasil-9, as the name suggests, contains 9 HPV VLPs. Other antibody-eliciting vaccines based on bacteriophage VLPs have been combined into multicomponent formulations and it appears that the only limitation to the number of individual VLPs included in one vaccine is how much protein and volume one can combine in a single injection [48]. The ease of generating a multicomponent vaccine means that vaccines can be made against pathogens for which multiple antigens must be targeted to elicit protection. This would be advantageous for Ct in particular, which may require multiple antigens to protect against the many bacterial serovars that can cause disease.

Vaccines that mediate protection only through antibodies generate responses that can be tailored to a specific antigen, and even to a specific epitope of that antigen. This provides a unique opportunity to elicit almost monoclonal-like responses to an epitope of interest. This is useful when targeting a so-called “cryptic epitope”, which is not normally immunogenic but could be protective if antibodies are produced by vaccination. This strategy has been employed in efforts to generate a “Pan-HPV” vaccine by targeting the highly conserved HPV L2 epitope described above [48]. Displaying a short, conserved epitope of L2 on the surface of a VLP vaccine platform generates an immunogen capable of eliciting specific antibody responses that protect against a broad range of HPV types [48]. Highly specific targeting of epitopes could also be useful if the protein antigen contains epitopes that act as “immunological decoys”. Avoiding targeting these epitopes could increase vaccine potency.

Vaccines developed to elicit antibodies against Ct present the opportunity for a simplified pre-clinical pipeline. Neutralization and opsonophagocytic killing of Ct can be tested without needing an animal model that fully recapitulates the pathogen-associated pathology present in humans. Indeed, HPV vaccines against L2 protein are tested in a mouse model of virus entry using a pseudovirus with a luciferase reporter system [56]. In this model, mice are vaccinated, challenged with pseudovirus, and then entry of the virus into cells in vivo can be assayed several days after infection with intravital imaging to detect expression of the luciferase reporter gene. Because HPV is host-species specific, similar to Ct, this sort of animal model is useful for assessing vaccine candidates that are only anticipated to block entry of the virus into host cells. In vitro assays, including neutralization assays and opsonization assays, are also relevant pre-clinical models for assessing antibody-only eliciting vaccines. We foresee the following pre-clinical pipeline for testing these types of vaccines: (1) demonstration of the ability of the vaccine to elicit high titer, long-lasting antibody responses in rodents, (2) demonstration of the elicited antibodies to either neutralize Ct infection in vitro AND/OR opsonophagocytic killing of Ct, (3) demonstration of prevention of early establishment of infection in an appropriate animal model, similar to the one described above for HPV, that recapitulates the early infection steps of Ct.

6.0. CONCLUSION

In this Perspective, we describe the immunologic basis of the successful HPV vaccine, namely that the HPV vaccine provides long-lasting, high titer, protective antibody in the genital tract, providing sterilizing immunity. We argue that the success of the VLP-based HPV vaccine and the challenges experienced by therapeutic HPV vaccine efforts, can inform Ct vaccine efforts. Whereas T cells play an important role in providing immunity to Ct during natural infection, we argue that using vaccine platforms that elicit high titer antibodies in the genital tract and eliciting antibodies to “cryptic epitopes” may be a promising vaccination strategy. Advantages of an antibody-eliciting vaccine strategy are the possibility of a single-dose vaccine, multivalent vaccine formulations to protect against multiple Ct serovars, and the opportunity for a simplified pre-clinical pipeline.

7.0. EXPERT COMMENTARY

The success of the HPV vaccine, in particular in inducing responses superior to those generated by natural infection, opens the door to the possibility that other sexually transmitted obligate intracellular pathogens could be targeted with a similar strategy. The key is to elicit the right antibody, in the right location, at high enough titers to protect. In the case of Ct, protection could be mediated by either/both (1) neutralization of the EBs such that they cannot establish a productive infection, either through prevention of adhesion or entry, and/or (2) opsonization of extracellular infectious EBs and increased clearance of the extracellular pathogen. Although natural infection or animal models of infection can provide clues to promising strategies for vaccines, as we know from HPV vaccine efforts targeting L2, it is often the case that the natural immune response is not necessarily the best response to mimic with a vaccine. This may be especially true in the case of Ct, for which the primary disease endpoint is caused by the cellular immune response to infection. Indeed, in animal models and humans, there is evidence of the harm caused by pathology-driving versus protective immune responses [33, 57, 58]. We propose that HPV provides a model for other obligate intracellular sexually transmitted pathogens, such as Ct, and provides evidence that a successful vaccine does not necessarily need to mimic the natural protective immune response to a pathogen.

The key to generating high titer, long lasting antibodies in response to intramuscular immunization without added adjuvant is the VLP platform. When other epitopes not included in the current HPV vaccines, like L2 peptides, are displayed on the surface of VLPs, these elicit high titer, long lasting antibody responses upon intramuscular immunization and provide protection against HPV pseudovirus infection in mice [48, 55, 59]. The success of VLPs as a vaccine platform is due to two features: size and geometry. Most VLPs are between 20–100nm which is the ideal size for uptake into antigen presenting cells and trafficking to the lymph nodes[60]. Their geometry, which is highly repetitive, rigid, and multivalent, presents antigen in an optimum format for strong B cell receptor signaling, leading to robust B cell activation and long lasting, high titer antibody responses [61]. There is growing interest in using VLPs derived from a variety of viruses as platforms to display heterologous antigens for vaccines. Displaying specific Ct antigens on the surface of VLPs or other multivalent vaccine platforms could be one approach for eliciting high titer, long-lasting antibody responses against Ct.

Choosing the correct antigenic epitopes can be challenging. However, basic science research investigating the entry mechanisms of pathogens can inform antibody-eliciting vaccine efforts. Indeed, experiments that have finely mapped and investigated entry mechanisms for HPV have informed next-generation HPV vaccines targeting the L2 protein and specific L2 peptides [62]. Choosing the right antigen epitopes may require detailed knowledge about the structure/function relationship of antigens of interest. In some cases, we can be guided by epitopes recognized by monoclonal antibodies with activity of interest (i.e. with neutralizing or opsonizing activity). In other cases, bioinformatics tools (such as surface exposure prediction tools) can be used to rationally select antigens to test. In some cases, looking for highly conserved, “cryptic epitopes”, similar to the L2 peptide for HPV, can lead to successful vaccines. For a complex pathogen like Ct, it may be necessary to include multiple antigen epitopes in a final vaccine to achieve full protection. We imagine that a successful antibody-eliciting vaccine against Ct would benefit from targeting multiple antigens involved in entry, adhesion, and opsonophagocytic killing of Ct. It is important to note that, although experiments describing the nature of the antigenicity of the HPV vaccine have been done in retrospect, these aspects of the vaccine were not critical to the approval of the vaccine nor the decision to move into human clinical trials. These decisions were made on the basis of strong evidence of protection in animal studies, in vitro neutralization assays, and the human trials.

Antibody is not necessary for natural immunity to Ct, but, as with HPV, it may be sufficient to protect if (1) the right antigen(s) is chosen, (2) high enough titers are elicited, (3) these antibodies are in the right anatomic location, (4) they are long lasting, (5) the antibody-mediated protection mechanisms we describe in this manuscript are effective in vivo, and (6) the endpoint of the vaccine trial is preventing infection rather than preventing disease sequelae. The Ct vaccine currently in Phase I clinical trials has been shown to elicit both strong T-cell and antibody responses to Ct MOMP [63, 64]. Several other experimental Ct vaccines that work by eliciting appropriate T-cell responses are in the pipeline [63, 64, 65, 66]. Clinical trials will inform whether these promising vaccine strategies are effective and safe. We propose a complementary approach for designing antibody-eliciting vaccines which may also prove fruitful against Ct given the tremendous success of the antibody-eliciting HPV vaccines at inducing sterilizing immunity in the genital tract. While we recognize that what worked for HPV may not necessarily work for Ct, we aim in this Perspective to encourage further discussion between HPV and Ct researchers about the similarities and differences between the two vaccine design efforts that might advance both fields.

8.0. FIVE-YEAR VIEW

In the next five years, we anticipate that recently developed tools for manipulating the Ct genome will open new opportunities to explore the role of specific Ct proteins in entry, establishment of infection in the host cell, pathogenesis, and immune evasion. Additionally, ongoing research investigating the immunopathology of Ct in animal models and humans will continue to differentiate protective versus pathology-driving immune responses. Together, this increase in our knowledge of basic Ct biology will provide new avenues to rationally design vaccines for Ct. With so much yet to understand about Ct pathogenesis, we are excited by the possibility that antibody-eliciting vaccines may yet play a role in protecting against this important human pathogen, just as they have for HPV.

9.0. KEY ISSUES.

• Ct is the most common bacterial sexually transmitted infection, and infection can lead to pelvic inflammatory disease and infertility in women

• The successful HPV vaccine provides protection by eliciting high-titer, long-lasting antibody responses that neutralize viral infection.

• HPV vaccines provide three lessons that can be applied to Ct vaccine efforts: (1) HPV vaccine has demonstrated that antibodies alone are sufficient to protect against an STI, (2) the successful generation of high levels of antibodies against HPV is attributed to the multivalent structure of HPV VLPs, and (3) there are major challenges in designing successful vaccines for inducing effector T cell responses against STI antigens in the genital tract.

• Although antibody is not necessary for natural immunity to Ct, antibody may be sufficient to protect if the right antigens are chosen, high enough titers are elicited, these antibodies are in the right anatomic location, the antibody is long-lasting, the antibody is functional in vivo, and if preventing infection is sufficient to prevent long-term sequelae.