Combined regulation of pro-inflammatory cytokines production by STAT3 and STAT5 in a model of B. pertussis infection of alveolar macrophages.

Bordetella pertussis is a highly contagious respiratory pathogen responsible for whooping-cough or pertussis. Despite high vaccination coverage worldwide, this gram-negative bacterium continues to spread among the population. B. pertussis is transmitted by aerosol droplets from an infected individual to a new host and will colonize its upper respiratory tract. Alveolar macrophages (AMs) are effector cells of the innate immune system that phagocytose B. pertussis and secrete both pro-inflammatory and antimicrobial mediators in the lungs. However, understanding their role in B. pertussis pathogenesis at the molecular level is hampered by the limited number of primary AMs that can be collected in vivo. In order to decipher the regulation of innate response induced by B. pertussis infection, we used for the first time self-renewing, non-transformed cells, called Max Planck Institute (MPI) cells, which are phenotypically and functionally very close to pulmonary AMs. Using optimized infection conditions, we characterized the entry and the clearance of B. pertussis within MPI macrophages. We showed that under these conditions, MPI cells exhibit a pro-inflammatory phenotype with the production of TNF, IL-1ß, IL-6 and MIP-2α, similarly to primary AMs purified from broncho-alveolar fluids of mice. In addition, we explored the yet uncharacterized role of the signal transduction activator of transcription (STAT) proteins family in the innate immune response to B. pertussis infection and showed for the first time the parallel regulation of pro-inflammatory cytokines by STAT3 and STAT5 in MPI macrophages infected by B. pertussis. Altogether, this work highlights the interest of using MPI cells for experiments optimization and preliminary data acquisition to understand B. pertussis interaction with AMs, and thus significantly reduce the number of animals to be sacrificed.

Live attenuated pertussis vaccine for prevention and treatment of allergic airway inflammation in mice.

Live attenuated vaccines often have beneficial non-specific effects, protecting against heterologous infectious and non-infectious diseases. We have developed a live attenuated pertussis vaccine, named BPZE1, currently in advanced clinical development. Here, we examined the prophylactic and therapeutic potential of its pertactin-deficient derivative BPZE1P in a mouse model of house dust mite (HDM)-induced allergic airway inflammation (AAI). BPZE1P was given nasally either before or after sensitization with HDM, followed by HDM challenge, or between two challenge episodes. Vaccination prior to sensitization reduced resistance in the airways, the numbers of infiltrating eosinophils and the concentrations of proinflammatory cytokines, such as IL-1α, IL-1ß and IL-33, in the lungs but had no effect on Th2 cytokine levels. BPZE1P also protected when delivered after sensitization or between two challenge episodes. However, in this case the levels of Th2 cytokines in the lung were decreased without significant effects on IL-1α, IL-1ß and IL-33 production. The vaccine restored lung function and decreased eosinophil influx in the lungs of HDM-treated mice. BPZE1P has a better take than BPZE1 in hosts vaccinated with acellular pertussis vaccines. Therefore, it has interesting potential as a preventive and therapeutic agent against AAI, even in acellular pertussis-vaccinated populations.

Live attenuated Bordetella pertussis vaccine candidate BPZE1 transiently protects against lethal pneumococcal disease in mice.

BPZE1 is a live attenuated vaccine against infection by Bordetella pertussis, the causative agent of whooping cough. It was previously shown that BPZE1 provides heterologous protection in mouse models of disease caused by unrelated pathogens, such as influenza virus and respiratory syncytial virus. Protection was also observed in mouse models of asthma and contact dermatitis. In this study, we demonstrate that BPZE1 also displays protection against an unrelated bacterial pathogen in a mouse model of invasive pneumococcal disease mediated by Streptococcus pneumoniae. While a single administration of BPZE1 provided no protection, two doses of 106 colony-forming units of BPZE1 given in a three-week interval protected against mortality, lung colonization and dissemination in both BALB/c and C57BL/6 mice. Unlike for the previously reported influenza challenge model, protection was short-lived, and waned within days after booster vaccination. Formaldehyde-killed BPZE1 protected only when administered following a live prime, indicating that priming requires live BPZE1 for protection. Protection against mortality was directly linked to substantially decreased bacterial dissemination in the blood and was lost in MyD88 knock-out mice, demonstrating the role of the innate immune system in the mechanism of protection. This is the first report on a heterologous protective effect of the live BPZE1 vaccine candidate against an unrelated bacterial infection.

Construction and evaluation of a pertactin-deficient live attenuated pertussis vaccine candidate BPZE1 derivative.

Pertussis, mainly caused by Bordetella pertussis, is a severe respiratory disease that can be fatal, especially in young infants. Vaccines, massively implemented since the middle of the last century, have substantially reduced the pertussis incidence, but have not been able to fully control the disease. One of the shortcomings of current pertussis vaccines is their inability to prevent infection by and transmission of B. pertussis, in contrast to immunity following natural infection. We have developed the live attenuated nasal vaccine BPZE1 and have shown that it prevents both disease and B. pertussis infection in preclinical models. This vaccine is now in clinical development. However, the initial clinical studies have suggested that vaccine take is hampered by pre-existing antibodies to pertactin. Here, we have constructed a pertactin-deficient BPZE1 derivative called BPZE1P in order to overcome this limitation. BPZE1P colonized the murine respiratory tract as efficiently as BPZE1 and induced antibodies at levels similar to those elicited by BPZE1. In the presence of pre-existing antibodies induced by acellular pertussis vaccination, BPZE1P colonized the mouse respiratory tract more efficiently than BPZE1. Both vaccines protected equally well the murine lungs and noses from challenge with laboratory and clinical strains of B. pertussis, including pertactin-deficient strains, against which current acellular pertussis vaccines are less efficient. BPZE1P may thus be an interesting alternative to BPZE1 to overcome vaccine take limitations due to pre-existing antibodies to pertactin.

Suppression of mucosal Th17 memory responses by acellular pertussis vaccines enhances nasal Bordetella pertussis carriage.

Pertussis has made a spectacular rebound in countries that have switched from whole-cell (wPV) to acellular pertussis vaccines (aPV). Here, we show that, unlike wPV, aPV, while protective against lung colonization by Bordetella pertussis (Bp), did not protect BALB/c mice from nasal colonization, but instead substantially prolonged nasal carriage. aPV prevented the natural induction of nasal interleukin-17 (IL-17)-producing and interferon-γ (IFN-γ)-producing CD103+ CD44+ CD69+ CD4+-resident memory T (TRM) cells. IL-17-deficient, but not IFN-γ-deficient, mice failed to clear nasal Bp, indicating a key role of IL-17+ TRM cells in the control of nasal infection. These cells appeared essential for neutrophil recruitment, crucial for clearance of Bp tightly bound to the nasal epithelium. Transfer of IL-17+ TRM cells from Bp-infected mice to IL-17-deficient mice resulted in neutrophil recruitment and protection against nasal colonization. Thus, aPV may have augmented the Bp reservoir by inhibiting natural TRM cell induction and neutrophil recruitment, thereby contributing to the pertussis resurgence.

Manufacture of a Stable Lyophilized Formulation of the Live Attenuated Pertussis Vaccine BPZE1.

Current pertussis vaccines protect against disease, but not against colonization by and transmission of Bordetella pertussis, whereas natural infection protects against both. The live attenuated vaccine BPZE1 was developed to mimic immunogenicity of natural infection without causing disease, and in preclinical models protected against pertussis disease and B. pertussis colonization after a single nasal administration. Phase 1 clinical studies showed that BPZE1 is safe and immunogenic in humans when administered as a liquid formulation, stored at ≤-70 °C. Although BPZE1 is stable for two years at ≤-70 °C, a lyophilized formulation stored at ≥5 °C is required for commercialization. The development of a BPZE1 drug product, filled and lyophilized directly in vials, showed that post-lyophilization survival of BPZE1 depended on the time of harvest, the lyophilization buffer, the time between harvest and lyophilization, as well as the lyophilization cycle. The animal component-free process, well defined in terms of harvest, processing and lyophilization, resulted in approximately 20% survival post-lyophilization. The resulting lyophilized drug product was stable for at least two years at -20 °C ± 10 °C, 5 °C ± 3 °C and 22.5 °C ± 2.5 °C and maintained its vaccine potency, as evaluated in a murine protection assay. This manufacturing process thus enables further clinical and commercial development of BPZE1.

Correction: Evaluation of inactivated Bordetella pertussis as a delivery system for the immunization of mice with Pneumococcal Surface Antigen A.

[This corrects the article DOI: 10.1371/journal.pone.0228055.].

Evaluation of inactivated Bordetella pertussis as a delivery system for the immunization of mice with Pneumococcal Surface Antigen A.

Pneumococcal Surface Protein A (PspA) has been successfully tested as vaccine candidate against Streptococcus pneumoniae infections. Vaccines able to induce PspA-specific antibodies and Th1 cytokines usually provide protection in mice. We have shown that the whole cell pertussis vaccine (wP) or components from acellular pertussis vaccines, such as Pertussis Toxin or Filamentous Hemagglutinin (FHA), are good adjuvants to PspA, suggesting that combined pertussis-PspA vaccines would be interesting strategies against the two infections. Here, we evaluated the potential of wP as a delivery vector to PspA. Bordetella pertussis strains producing a PspA from clade 4 (PspA4Pro) fused to the N-terminal region of FHA (Fha44) were constructed and inactivated with formaldehyde for the production of wPPspA4Pro. Subcutaneous immunization of mice with wPPspA4Pro induced low levels of anti-PspA4 IgG, even after 3 doses, and did not protect against a lethal pneumococcal challenge. Prime-boost strategies using wPPspA4Pro and PspA4Pro showed that there was no advantage in using the wPPspA4Pro vaccine. Immunization of mice with purified PspA4Pro induced higher levels of antibodies and protection against pneumococcal infection than the prime-boost strategies. Finally, purified Fha44:PspA4Pro induced high levels of anti-PspA4Pro IgG, but no protection, suggesting that the antibodies induced by the fusion protein were not directed to protective epitopes.

Evaluation of inactivated Bordetella pertussis as a delivery system for the immunization of mice with Pneumococcal Surface Antigen A

Pneumococcal Surface Protein A (PspA) has been successfully tested as vaccine candidate against Streptococcus pneumoniae infections. Vaccines able to induce PspA-specific antibodies and Th1 cytokines usually provide protection in mice. We have shown that the whole cell pertussis vaccine (wP) or components from acellular pertussis vaccines, such as Pertussis Toxin or Filamentous Hemagglutinin (FHA), are good adjuvants to PspA, suggesting that combined pertussis-PspA vaccines would be interesting strategies against the two infections. Here, we evaluated the potential of wP as a delivery vector to PspA. Bordetella pertussis strains producing a PspA from clade 4 (PspA4Pro) fused to the N-terminal region of FHA (Fha44) were constructed and inactivated with formaldehyde for the production of wPPspA4Pro. Subcutaneous immunization of mice with wPPspA4Pro induced low levels of anti-PspA4 IgG, even after 3 doses, and did not protect against a lethal pneumococcal challenge. Prime-boost strategies using wPPspA4Pro and PspA4Pro showed that there was no advantage in using the wPPspA4Pro vaccine. Immunization of mice with purified PspA4Pro induced higher levels of antibodies and protection against pneumococcal infection than the prime-boost strategies. Finally, purified Fha44:PspA4Pro induced high levels of anti-PspA4Pro IgG, but no protection, suggesting that the antibodies induced by the fusion protein were not directed to protective epitopes.

Evaluation of inactivated Bordetella pertussis as a delivery system for the immunization of mice with Pneumococcal Surface Antigen A

Pneumococcal Surface Protein A (PspA) has been successfully tested as vaccine candidate against Streptococcus pneumoniae infections. Vaccines able to induce PspA-specific antibodies and Th1 cytokines usually provide protection in mice. We have shown that the whole cell pertussis vaccine (wP) or components from acellular pertussis vaccines, such as Pertussis Toxin or Filamentous Hemagglutinin (FHA), are good adjuvants to PspA, suggesting that combined pertussis-PspA vaccines would be interesting strategies against the two infections. Here, we evaluated the potential of wP as a delivery vector to PspA. Bordetella pertussis strains producing a PspA from clade 4 (PspA4Pro) fused to the N-terminal region of FHA (Fha44) were constructed and inactivated with formaldehyde for the production of wPPspA4Pro. Subcutaneous immunization of mice with wPPspA4Pro induced low levels of anti-PspA4 IgG, even after 3 doses, and did not protect against a lethal pneumococcal challenge. Prime-boost strategies using wPPspA4Pro and PspA4Pro showed that there was no advantage in using the wPPspA4Pro vaccine. Immunization of mice with purified PspA4Pro induced higher levels of antibodies and protection against pneumococcal infection than the prime-boost strategies. Finally, purified Fha44:PspA4Pro induced high levels of anti-PspA4Pro IgG, but no protection, suggesting that the antibodies induced by the fusion protein were not directed to protective epitopes.

Early Protection against Pertussis Induced by Live Attenuated Bordetella pertussis BPZE1 Depends on TLR4.

Pertussis is a severe respiratory disease mainly caused by Bordetella pertussis Despite wide global vaccination coverage with efficacious pertussis vaccines, it remains one of the least well-controlled vaccine-preventable diseases, illustrating the shortcomings of the current vaccines. We have developed the live attenuated nasal pertussis vaccine BPZE1, currently undergoing clinical evaluation in human phase 2 trials. We have previously shown that in mice, BPZE1 provides strong and long-lasting protection against B. pertussis challenge by inducing potent Ab and T cell responses as well as secretory IgA and IL-17-producing resident memory T lymphocytes in the nasal cavity. In this study, we show that BPZE1 induces protection in mice against B. pertussis within days after vaccination, at a time when Ab and T cell responses were not detectable. Early protection was independent of T and B cell responses, as demonstrated by the use of SCID mice. Instead, it was due to TLR4-dependent signaling through the MyD88-dependent pathway of the innate immune response, as demonstrated in experiments with TLR4-deficient and MyD88-knockout mice. TLR2-dependent signaling did not play a major role in early protection. In addition, this study also shows that even at high doses, BPZE1 is safe in the severely immunocompromised MyD88-deficient mice, whereas virulent B. pertussis caused a severe pathological condition and death in these mice, even at a low dose. Finally, coadministration of virulent B. pertussis with BPZE1 did not cause exacerbated outgrowth of the virulent strain, thereby adding to the safety profile of this live vaccine candidate.

Intrinsic Antibacterial Activity of Nanoparticles Made of ß-Cyclodextrins Potentiates Their Effect as Drug Nanocarriers against Tuberculosis.

Multi-drug-resistant tuberculosis (TB) is a major public health problem, concerning about half a million cases each year. Patients hardly adhere to the current strict treatment consisting of more than 10 000 tablets over a 2-year period. There is a clear need for efficient and better formulated medications. We have previously shown that nanoparticles made of cross-linked poly-ß-cyclodextrins (pßCD) are efficient vehicles for pulmonary delivery of powerful combinations of anti-TB drugs. Here, we report that in addition to being efficient drug carriers, pßCD nanoparticles are endowed with intrinsic antibacterial properties. Empty pßCD nanoparticles are able to impair Mycobacterium tuberculosis (Mtb) establishment after pulmonary administration in mice. pßCD hamper colonization of macrophages by Mtb by interfering with lipid rafts, without inducing toxicity. Moreover, pßCD provoke macrophage apoptosis, leading to depletion of infected cells, thus creating a lung microenvironment detrimental to Mtb persistence. Taken together, our results suggest that pßCD nanoparticles loaded or not with antibiotics have an antibacterial action on their own and could be used as a carrier in drug regimen formulations effective against TB.

Distinct virulence ranges for infection of mice by Bordetella pertussis revealed by engineering of the sensor-kinase BvgS.

The whooping cough agent Bordetella pertussis coordinately regulates the expression of its virulence factors with the two-component system BvgAS. In laboratory conditions, specific chemical modulators are used to trigger phenotypic modulation of B. pertussis from its default virulent Bvg+ phase to avirulent Bvg- or intermediate Bvgi phases, in which no virulence factors or only a subset of them are produced, respectively. Whether phenotypic modulation occurs in the host remains unknown. In this work, recombinant B. pertussis strains harboring BvgS variants were tested in a mouse model of infection and analyzed using transcriptomic approaches. Recombinant BP-BvgΔ65, which is in the Bvgi phase by default and can be up-modulated to the Bvg+ phase in vitro, could colonize the mouse nose but was rapidly cleared from the lungs, while Bvg+-phase strains colonized both organs for up to four weeks. These results indicated that phenotypic modulation, which might have restored the full virulence capability of BP-BvgΔ65, does not occur in mice or is temporally or spatially restricted and has no effect in those conditions. Transcriptomic analyses of this and other recombinant Bvgi and Bvg+-phase strains revealed that two distinct ranges of virulence gene expression allow colonization of the mouse nose and lungs, respectively. We also showed that a recombinant strain expressing moderately lower levels of the virulence genes than its wild type parent was as efficient at colonizing both organs. Altogether, genetic modifications of BvgS generate a range of phenotypic phases, which are useful tools to decipher host-pathogen interactions.

IL-17-dependent SIgA-mediated protection against nasal Bordetella pertussis infection by live attenuated BPZE1 vaccine.

BPZE1 is a live attenuated Bordetella pertussis vaccine for nasal administration to mimic the natural route of infection. Here, we studied the mechanism of BPZE1-induced immunity in the murine nasal cavity in contrast to acellular vaccine (aPV), although both vaccines protected against lung colonization. Transfer of splenocytes or serum from BPZE1-vaccinated or aPV-vaccinated mice protected naïve mice against lung colonization but not against nasal colonization. However, transfer of nasal washes from BPZE1-vaccinated mice resulted in protection against nasal colonization, which was lost in IgA-deficient or poly-Ig receptor-deficient mice, indicating that it depends on secretory IgA (SIgA) induction induced in the nose. BPZE1-induced protection against nasal colonization was long-lived despite the relatively rapid decay of SIgA, indicating a potent BPZE1-induced local memory response, likely due to CD4+ tissue-resident memory T cells induced in the nose by BPZE1. These cells produced interleukin-17 (IL-17), known to be important for SIgA secretion. Furthermore, BPZE1 failed to protect Il17-/- mice against nasal colonization by B. pertussis and induced only background levels of nasal SIgA. Thus, our results show important differences in the protective mechanism between the upper and the lower murine respiratory tract and demonstrate an IL-17-dependent SIgA-mediated mechanism of BPZE1-induced protection against B. pertussis nasopharyngeal colonization.

Construction and evaluation of Bordetella pertussis live attenuated vaccine strain BPZE1 producing Fim3.

Pertussis or whooping cough is currently the most prevalent vaccine-preventable childhood disease despite >85% global vaccination coverage. In recent years incidence has greatly increased in several high-income countries that have switched from the first-generation, whole-cell vaccine to the newer acellular vaccines, calling for improved vaccination strategies with better vaccines. We have developed a live attenuated pertussis vaccine candidate, called BPZE1, which is currently in clinical development. Unlike other pertussis vaccines, BPZE1 has been shown to provide strong protection against infection by the causative agent of pertussis, Bordetella pertussis, in non-human primates. BPZE1 is a derivative of the B. pertussis strain Tohama I, which produces serotype 2 (Fim2) but not serotype 3 fimbriae (Fim3). As immune responses to fimbriae are likely to contribute to protection, we constructed a BPZE1 derivative, called BPZE1f3, that produces both serotypes of fimbriae. Whereas nasal vaccination of mice with BPZE1 induced antibodies to Fim2 but not to Fim3, vaccination with BPZE1f3 elicited antibodies to both Fim2 and Fim3 at approximately the same level. In mice, both BPZE1 and BPZE1f3 provided equal levels of protection against clinical isolates that either produce Fim2 alone, both Fim2 and Fim3, or no fimbriae. However, vaccination with BPZE1f3 provided significantly stronger protection against Fim3-only producing B. pertussis than vaccination with BPZE1, indicating that immune responses to fimbriae contribute to serotype-specific protection against B. pertussis infection.

Mycobacterium tuberculosis Controls Phagosomal Acidification by Targeting CISH-Mediated Signaling.

Pathogens have evolved a range of mechanisms to counteract host defenses, notably to survive harsh acidic conditions in phagosomes. In the case of Mycobacterium tuberculosis, it has been shown that regulation of phagosome acidification could be achieved by interfering with the retention of the V-ATPase complexes at the vacuole. Here, we present evidence that M. tuberculosis resorts to yet another strategy to control phagosomal acidification, interfering with host suppressor of cytokine signaling (SOCS) protein functions. More precisely, we show that infection of macrophages with M. tuberculosis leads to granulocyte-macrophage colony-stimulating factor (GM-CSF) secretion, inducing STAT5-mediated expression of cytokine-inducible SH2-containing protein (CISH), which selectively targets the V-ATPase catalytic subunit A for ubiquitination and degradation by the proteasome. Consistently, we show that inhibition of CISH expression leads to reduced replication of M. tuberculosis in macrophages. Our findings further broaden the molecular understanding of mechanisms deployed by bacteria to survive.

Live Attenuated Pertussis Vaccine BPZE1 Protects Baboons Against Bordetella pertussis Disease and Infection.

Evidence suggests that the resurgence of pertussis in many industrialized countries may result from the failure of current vaccines to prevent nasopharyngeal colonization by Bordetella pertussis, the principal causative agent of whooping cough. Here, we used a baboon model to test the protective potential of the novel, live attenuated pertussis vaccine candidate BPZE1. A single intranasal/intratracheal inoculation of juvenile baboons with BPZE1 resulted in transient nasopharyngeal colonization and induction of immunoglobulin G and immunoglobulin A to all antigens tested, while causing no adverse symptoms or leukocytosis. When BPZE1-vaccinated baboons were challenged with a high dose of a highly virulent B. pertussis isolate, they were fully protected against disease, whereas naive baboons developed illness (with 1 death) and leukocytosis. Total postchallenge nasopharyngeal virulent bacterial burden of vaccinated animals was substantially reduced (0.002%) compared to naive controls, providing promising evidence in nonhuman primates that BPZE1 protects against both pertussis disease and B. pertussis infection.

LppM impact on the colonization of macrophages by Mycobacterium tuberculosis.

Mycobacterium tuberculosis produces several bacterial effectors impacting the colonization of phagocytes. Here, we report that the putative lipoprotein LppM hinders phagocytosis by macrophages in a toll-like receptor 2-dependent manner. Moreover, recombinant LppM is able to functionally complement the phenotype of the mutant, when exogenously added during macrophage infection. LppM is also implicated in the phagosomal maturation, as a lppM deletion mutant is more easily addressed towards the acidified compartments of the macrophage than its isogenic parental strain. In addition, this mutant was affected in its ability to induce the secretion of pro-inflammatory chemokines, interferon-gamma-inducible protein-10, monocyte chemoattractant protein-1 and macrophage inflammatory protein-1α. Thus, our results describe a new mycobacterial protein involved in the early trafficking of the tubercle bacillus and its manipulation of the host immune response.

STAT3 Represses Nitric Oxide Synthesis in Human Macrophages upon Mycobacterium tuberculosis Infection.

Mycobacterium tuberculosis is a successful intracellular pathogen. Numerous host innate immune responses signaling pathways are induced upon mycobacterium invasion, however their impact on M. tuberculosis replication is not fully understood. Here we reinvestigate the role of STAT3 specifically inside human macrophages shortly after M. tuberculosis uptake. We first show that STAT3 activation is mediated by IL-10 and occurs in M. tuberculosis infected cells as well as in bystander non-colonized cells. STAT3 activation results in the inhibition of IL-6, TNF-α, IFN-γ and MIP-1ß. We further demonstrate that STAT3 represses iNOS expression and NO synthesis. Accordingly, the inhibition of STAT3 is detrimental for M. tuberculosis intracellular replication. Our study thus points out STAT3 as a key host factor for M. tuberculosis intracellular establishment in the early stages of macrophage infection.

HBHA vaccination may require both Th1 and Th17 immune responses to protect mice against tuberculosis.

Almost one century after the discovery of the BCG vaccine, tuberculosis remains a major cause of global mortality and morbidity, emphasizing the urgent need to design more efficient vaccines. The heparin-binding haemagglutinin (HBHA) appears to be a promising vaccine candidate, as it was shown to afford protection to mice against a challenge infection with Mycobacterium tuberculosis when combined with the strong adjuvant DDA/MPL (dimethyldioctadecyl-ammonium bromide/monophosphoryl lipid A), a TLR4 ligand. In this study, we investigated the immunological response and protection of mice immunized with HBHA formulated in lipid-containing nanoparticles and adjuvanted with CpG, a TLR9 ligand. Subcutaneous immunization with this HBHA formulation led to a marked Th1 response, characterized by high IFN-γ levels, but no significant IL-17 production, both in spleen and lung, in contrast to DDA/MPL MPL-formulated HBHA, which induced both IFN-γ and IL-17. This cytokine profile was also observed in BCG-primed mice and persisted after M. tuberculosis infection. No significant protection was obtained against challenge infection after vaccination with the nanoparticle-CpG formulation, and this was associated with a failure to mount a memory immune response. These results suggest the importance of both Th1 and Th17 immune responses for vaccine-induced immunity.