The role of antigen availability during B cell induction and its effect on sustained memory and antibody production after infection and vaccination-lessons learned from the SARS-CoV-2 pandemic.

The importance of antibodies, particularly neutralizing antibodies, has been known for decades. When examining the immune responses against a pathogen after a vaccination or infection it is easier to measure the levels of antigen-specific antibodies than the T-cell response, but it does not give the whole picture. The levels of neutralizing antibodies are harder to determine but give a better indication of the quality of the antibody response. The induction of long-lived antibody-secreting plasma cells is crucial for a persistent humoral immune response, which has been shown for example after vaccination with the vaccinia vaccine, where antibody levels have been shown to persist for decades. With the SARS-CoV-2 pandemic ravaging the world for the past years and the monumental effort in designing and releasing novel vaccines against the virus, much effort has been put into analysing the quantity, quality, and persistence of antibody responses.

A comparative study of adjuvants effects on neonatal plasma cell survival niche in bone marrow and persistence of humoral immune responses.

The neonatal immune system is distinct from the immune system of older individuals rendering neonates vulnerable to infections and poor responders to vaccination. Adjuvants can be used as tools to enhance immune responses to co-administered antigens. Antibody (Ab) persistence is mediated by long-lived plasma cells that reside in specialized survival niches in the bone marrow, and transient Ab responses in early life have been associated with decreased survival of plasma cells, possibly due to lack of survival factors. Various cells can secrete these factors and which cells are the main producers is still up for debate, especially in early life where this has not been fully addressed. The receptor BCMA and its ligand APRIL have been shown to be important in the maintenance of plasma cells and Abs. Herein, we assessed age-dependent maturation of a broad range of bone marrow accessory cells and their expression of the survival factors APRIL and IL-6. Furthermore, we performed a comparative analysis of the potential of 5 different adjuvants; LT-K63, mmCT, MF59, IC31 and alum, to enhance expression of survival factors and BCMA following immunization of neonatal mice with tetanus toxoid (TT) vaccine. We found that APRIL expression was reduced in the bone marrow of young mice whereas IL-6 expression was higher. Eosinophils, macrophages, megakaryocytes, monocytes and lymphocytes were important secretors of survival factors in early life but undefined cells also constituted a large fraction of secretors. Immunization and adjuvants enhanced APRIL expression but decreased IL-6 expression in bone marrow cells early after immunization. Furthermore, neonatal immunization with adjuvants enhanced the proportion of plasmablasts and plasma cells that expressed BCMA both in spleen and bone marrow. Enhanced BCMA expression correlated with enhanced vaccine-specific humoral responses, even though the effect of alum on BCMA was less pronounced than those of the other adjuvants at later time points. We propose that low APRIL expression in bone marrow as well as low BCMA expression of plasmablasts/plasma cells in early life together cause transient Ab responses and could represent targets to be triggered by vaccine adjuvants to induce persistent humoral immune responses in this age group.

The adjuvants dmLT and mmCT enhance humoral immune responses to a pneumococcal conjugate vaccine after both parenteral or mucosal immunization of neonatal mice.

Immaturity of the neonatal immune system contributes to increased susceptibility to infectious diseases and poor vaccine responses. Therefore, better strategies for early life vaccination are needed. Adjuvants can enhance the magnitude and duration of immune responses. In this study we assessed the effects of the adjuvants dmLT and mmCT and different immunization routes, subcutaneous (s.c.) and intranasal (i.n.), on neonatal immune response to a pneumococcal conjugate vaccine Pn1-CRM197. Pn1-specific antibody (Ab) levels of neonatal mice immunized with Pn1-CRM197 alone were low. The adjuvants enhanced IgG Ab responses up to 8 weeks after immunization, more after s.c. than i.n. immunization. On the contrary, i.n. immunization with either adjuvant enhanced serum and salivary IgA levels more than s.c. immunization. In addition, both dmLT and mmCT enhanced germinal center formation and accordingly, dmLT and mmCT enhanced the induction and persistence of Pn1-specific IgG+ Ab-secreting cells (ASCs) in spleen and bone marrow (BM), irrespective of the immunization route. Furthermore, i.n. immunization enhanced Pn1-specific IgA+ ASCs in BM more than s.c. immunizatiofimmu.2022.1078904n. However, a higher i.n. dose of the Pn1-CRM197 was needed to achieve IgG response comparable to that elicited by s.c. immunization with either adjuvant. We conclude that dmLT and mmCT enhance both induction and persistence of the neonatal immune response to the vaccine Pn1-CRM197, following mucosal or parenteral immunization. This indicates that dmLT and mmCT are promising adjuvants for developing safe and effective early life vaccination strategies.

LT-K63 Enhances B Cell Activation and Survival Factors in Neonatal Mice That Translates Into Long-Lived Humoral Immunity.

Adjuvants enhance magnitude and duration of immune responses induced by vaccines. In this study we assessed in neonatal mice if and how the adjuvant LT-K63 given with a pneumococcal conjugate vaccine, Pnc1-TT, could affect the expression of tumor necrosis factor receptor (TNF-R) superfamily members, known to be involved in the initiation and maintenance of antibody responses; B cell activating factor receptor (BAFF-R) and B cell maturation antigen (BCMA) and their ligands, BAFF, and a proliferation inducing ligand (APRIL). Initially we assessed the maturation status of different B cell populations and their expression of BAFF-R and BCMA. Neonatal mice had dramatically fewer B cells than adult mice and the composition of different subsets within the B cell pool differed greatly. Proportionally newly formed B cells were most abundant, but they had diminished BAFF-R expression which could explain low proportions of marginal zone and follicular B cells observed. Limited BCMA expression was also detected in neonatal pre-plasmablasts/plasmablasts. LT-K63 enhanced vaccine-induced BAFF-R expression in splenic marginal zone, follicular and newly formed B cells, leading to increased plasmablast/plasma cells, and their enhanced expression of BCMA in spleen and bone marrow. Additionally, the induction of BAFF and APRIL expression occurred early in neonatal mice immunized with Pnc1-TT either with or without LT-K63. However, BAFF+ and APRIL+ cells in spleens were maintained at a higher level in mice that received the adjuvant. Furthermore, the early increase of APRIL+ cells in bone marrow was more profound in mice immunized with vaccine and adjuvant. Finally, we assessed, for the first time in neonatal mice, accessory cells of the plasma cell niche in bone marrow and their secretion of APRIL. We found that LT-K63 enhanced the frequency and APRIL expression of eosinophils, macrophages, and megakaryocytes, which likely contributed to plasma cell survival, even though APRIL+ cells showed a fast decline. All this was associated with enhanced, sustained vaccine-specific antibody-secreting cells in bone marrow and persisting vaccine-specific serum antibodies. Our study sheds light on the mechanisms behind the adjuvanticity of LT-K63 and identifies molecular pathways that should be triggered by vaccine adjuvants to induce sustained humoral immunity in early life.

Short Vi-polysaccharide abrogates T-independent immune response and hyporesponsiveness elicited by long Vi-CRM197 conjugate vaccine.

Polysaccharide-protein conjugates have been developed to overcome the T-independent response, hyporesponsiveness to repeated vaccination, and poor immunogenicity in infants of polysaccharides. To address the impact of polysaccharide length, typhoid conjugates made with short- and long-chain fractions of Vi polysaccharide with average sizes of 9.5, 22.8, 42.7, 82.0, and 165 kDa were compared. Long-chain-conjugated Vi (165 kDa) induced a response in both wild-type and T cell-deficient mice, suggesting that it maintains a T-independent response. In marked contrast, short-chain Vi (9.5 to 42.7 kDa) conjugates induced a response in wild-type mice but not in T cell-deficient mice, suggesting that the response is dependent on T cell help. Mechanistically, this was explained in neonatal mice, in which long-chain, but not short-chain, Vi conjugate induced late apoptosis of Vi-specific B cells in spleen and early depletion of Vi-specific B cells in bone marrow, resulting in hyporesponsiveness and lack of long-term persistence of Vi-specific IgG in serum and IgG+ antibody-secreting cells in bone marrow. We conclude that while conjugation of long-chain Vi generates T-dependent antigens, the conjugates also retain T-independent properties, leading to detrimental effects on immune responses. The data reported here may explain some inconsistencies observed in clinical trials and help guide the design of effective conjugate vaccines.

Adjuvants Enhance the Induction of Germinal Center and Antibody Secreting Cells in Spleen and Their Persistence in Bone Marrow of Neonatal Mice.

Immaturity of the immune system contributes to poor vaccine responses in early life. Germinal center (GC) activation is limited due to poorly developed follicular dendritic cells (FDC), causing generation of few antibody-secreting cells (ASCs) with limited survival and transient antibody responses. Herein, we compared the potential of five adjuvants, namely LT-K63, mmCT, MF59, IC31, and alum to overcome limitations of the neonatal immune system and to enhance and prolong responses of neonatal mice to a pneumococcal conjugate vaccine Pnc1-TT. The adjuvants LT-K63, mmCT, MF59, and IC31 significantly enhanced GC formation and FDC maturation in neonatal mice when co-administered with Pnc1-TT. This enhanced GC induction correlated with significantly enhanced vaccine-specific ASCs by LT-K63, mmCT, and MF59 in spleen 14 days after immunization. Furthermore, mmCT, MF59, and IC31 prolonged the induction of vaccine-specific ASCs in spleen and increased their persistence in bone marrow up to 9 weeks after immunization, as previously shown for LT-K63. Accordingly, serum Abs persisted above protective levels against pneumococcal bacteremia and pneumonia. In contrast, alum only enhanced the primary induction of vaccine-specific IgG Abs, which was transient. Our comparative study demonstrated that, in contrast to alum, LT-K63, mmCT, MF59, and IC31 can overcome limitations of the neonatal immune system and enhance both induction and persistence of protective immune response when administered with Pnc1-TT. These adjuvants are promising candidates for early life vaccination.

Natural killer cells play an essential role in resolution of antigen-induced inflammation in mice.

This study examined whether NK cells are important for resolution of antigen-induced inflammation. C57BL/6 mice were immunized twice with methylated BSA (mBSA) and inflammation induced by intraperitoneal injection of mBSA. Mice were injected intravenously with anti-asialo GM1 (αASGM1) or a control antibody 24h prior to peritonitis induction and peritoneal exudate collected at different time points. Expression of surface molecules and apoptosis on peritoneal cells was determined by flow cytometry and concentration of chemokines, cytokines, soluble cytokine receptors and lipid mediators by ELISA and LC-MS/MS. Apoptosis in parathymic lymph nodes and spleens was determined by TUNEL staining. Mice administered αASGM1 had lower peritoneal NK cell numbers and a higher number of peritoneal neutrophils 12h after induction of inflammation than control mice. The number of neutrophils was still high in the αASGM1 treated mice when their number had returned to baseline levels in the control mice, 48h after induction of inflammation. Peritoneal concentrations of the neutrophil regulators G-CSF and IL-12p40 were higher at 12h in the αASGM1 treated mice than in the control mice, whereas concentrations of lipid mediators implicated in resolution of inflammation, i.e. LXA4 and PGE2, were lower. Reduced apoptosis was detected in peritoneal neutrophils as well as in draining lymph nodes and spleens from the αASGM1 treated mice compared with that in the control mice. In addition, αASGM1 treated mice had lower number of peritoneal NK cells expressing NKp46 and NKG2D, receptors implicated in NK cell-induced neutrophil apoptosis. Furthermore, αASGM1 treatment completely blocked the increase in CD27+ NK cells that occurred in control mice following induction of inflammation, but CD27+ NK cells have been suggested to have a regulatory role. These results indicate a crucial role for NK cells in resolution of antigen-induced inflammation and suggest their importance in tempering neutrophil recruitment and maintaining neutrophil apoptosis.

Pneumococcal polysaccharide abrogates conjugate-induced germinal center reaction and depletes antibody secreting cell pool, causing hyporesponsiveness.

BACKGROUND: Plain pneumococcal polysaccharide (PPS) booster administered during second year of life has been shown to cause hyporesponsiveness. We assessed the effects of PPS booster on splenic memory B cell responses and persistence of PPS-specific long-lived plasma cells in the bone marrow (BM). METHODS: Neonatal mice were primed subcutanously (s.c.) or intranasally (i.n.) with pneumococcal conjugate (Pnc1-TT) and the adjuvant LT-K63, and boosted with PPS+LT-K63 or saline 1, 2 or 3 times with 16 day intervals. Seven days after each booster, spleens were removed, germinal centers (GC), IgM(+), IgG(+) follicles and PPS-specific antibody secreting cells (AbSC) in spleen and BM enumerated. RESULTS: PPS booster s.c., but not i.n., compromised the Pnc1-TT-induced PPS-specific Abs by abrogating the Pnc1-TT-induced GC reaction and depleting PPS-specific AbSCs in spleen and limiting their homing to the BM. There was no difference in the frequency of PPS-specific AbSCs in spleen and BM between mice that received 1, 2 or 3 PPS boosters s.c.. Repeated PPS+LT-K63 booster i.n. reduced the frequency of PPS-specific IgG(+) AbSCs in BM. CONCLUSIONS: PPS booster-induced hyporesponsiveness is caused by abrogation of conjugate-induced GC reaction and depletion of PPS-specific IgG(+) AbSCs resulting in no homing of new PPS-specific long-lived plasma cells to the BM or survival. These results should be taken into account in design of vaccination schedules where polysaccharides are being considered.

The adjuvant LT-K63 can restore delayed maturation of follicular dendritic cells and poor persistence of both protein- and polysaccharide-specific antibody-secreting cells in neonatal mice.

Ab responses in early life are low and short-lived; therefore, induction of protective immunity requires repeated vaccinations. One of the major limitations in early-life immunity is delayed maturation of follicular dendritic cells (FDCs), which play a central role in mediating the germinal center (GC) reaction leading to production of Ab-secreting cells (AbSCs). We assessed whether a nontoxic mutant of Escherichia coli heat-labile enterotoxin (LT-K63) and CpG1826 as model adjuvants could accelerate FDC maturation and immune response in neonatal mice, using a pneumococcal polysaccharide of serotype 1 conjugated to tetanus toxoid (Pnc1-TT) as a model vaccine. In neonatal NMRI mice, a single dose of Pnc1-TT coadministered with LT-K63 enhanced Pnc1-TT-induced GC reaction. In contrast, CpG1826 had no effect. Accordingly, LT-K63, but not CpG1826, accelerated the maturation of FDC networks, detected by FDC-M2(+) staining, characteristic for adult-like FDCs. This coincided with migration of MOMA-1(+) macrophages into the GCs that can enhance GC reaction and B cell activation. The FDC-M2(+) FDC networks colocalized with enhanced expression of TNF-α, which is critical for the maintenance of mature FDCs and is poorly expressed in neonates. The accelerated maturation of FDC networks correlated with increased frequency and prolonged persistence of polysaccharide- and protein-specific IgG(+) AbSCs in spleen and bone marrow. Our data show for the first time, to our knowledge, that an adjuvant (LT-K63) can overcome delayed maturation of FDCs in neonates, enhance the GC reaction, and prolong the persistence of vaccine-specific AbSCs in the BM. These properties are attractive for parenteral vaccination in early life.

Hyporesponsiveness following booster immunization with bacterial polysaccharides is caused by apoptosis of memory B cells.

BACKGROUND: Repeated immunizations with polysaccharide (PS) vaccines cause hyporesponsiveness through undefined mechanisms. We assessed the effects of a PS booster on immune responses, frequency, and survival of PS-specific B-cell subpopulations in spleen and bone marrow. METHODS: Neonatal mice were primed with meningococcus serotype C (MenC) conjugate MenC-CRM(197)+CpG1826, boosted with MenC-CRM(197), MenC-PS, or saline; subsequently, bromodeoxyuridine (BrdU) was injected daily intraperitoneally. MenC-PS-specific cells were labeled with fluorescent MenC-PS and phenotyped by flow cytometry. RESULTS: After MenC-PS booster, proliferating (BrdU(+)) MenC-PS-specific naive B cells (CD138(-)/B220(+); P = .0003) and plasma cells (CD138(+)/B220(-); P = .0002) in spleen were fewer than after saline booster. BrdU(+) MenC-PS-specific plasma cells were also reduced in bone marrow (P = .0308). Compared to saline, MenC-PS booster reduced BrdU(+) IgG(+) MenC-PS-specific B cells in spleen (P = .0002). Twelve hours after the MenC-PS booster, an increased frequency of apoptotic (AnnexinV(+)) MenC-PS-specific B cells in spleen was observed compared with MenC-CRM(197) (P = .0286) or saline (P = .001) boosters. CONCLUSIONS: We demonstrated that the MenC-PS booster significantly reduced the frequency of newly activated MenC-PS-specific B cells-mostly switched IgG(+) memory cells-by driving them into apoptosis. It shows directly that apoptosis of PS-specific memory cells is the cause of PS-induced hyporesponsiveness. These results should be taken into account prior to consideration of the use of PS vaccines.

Competition for FcRn-mediated transport gives rise to short half-life of human IgG3 and offers therapeutic potential.

Human IgG3 displays the strongest effector functions of all IgG subclasses but has a short half-life for unresolved reasons. Here we show that IgG3 binds to IgG-salvage receptor (FcRn), but that FcRn-mediated transport and rescue of IgG3 is inhibited in the presence of IgG1 due to intracellular competition between IgG1 and IgG3. We reveal that this occurs because of a single amino acid difference at position 435, where IgG3 has an arginine instead of the histidine found in all other IgG subclasses. While the presence of R435 in IgG increases binding to FcRn at neutral pH, it decreases binding at acidic pH, affecting the rescue efficiency-but only in the presence of H435-IgG. Importantly, we show that in humans the half-life of the H435-containing IgG3 allotype is comparable to IgG1. H435-IgG3 also gave enhanced protection against a pneumococcal challenge in mice, demonstrating H435-IgG3 to be a candidate for monoclonal antibody therapies.

Concomitant administration of Mycobacterium bovis BCG with the meningococcal C conjugate vaccine to neonatal mice enhances antibody response and protective efficacy.

Mycobacterium bovis BCG is administered to human neonates in many countries worldwide. The objective of the study was to assess if BCG could act as an adjuvant for polysaccharide-protein conjugate vaccines in newborns and thereby induce protective immunity against encapsulated bacteria in early infancy when susceptibility is high. We assessed whether BCG could enhance immune responses to a meningococcal C (MenC) conjugate vaccine, MenC-CRM(197), in mice primed as neonates, broaden the antibody response from a dominant IgG1 toward a mixed IgG1 and IgG2a/IgG2b response, and increase protective efficacy, as measured by serum bactericidal activity (SBA). Two-week-old mice were primed subcutaneously (s.c.) with MenC-CRM(197). BCG was administered concomitantly, a day or a week before MenC-CRM(197). An adjuvant effect of BCG was observed only when it was given concomitantly with MenC-CRM(197), with increased IgG response (P = 0.002) and SBA (8-fold) after a second immunization with MenC-CRM(197) without BCG, indicating increased T-cell help. In neonatal mice (1 week old) primed s.c. with MenC-CRM(197) together with BCG, MenC-polysaccharide (PS)-specific IgG was enhanced compared to MenC-CRM(197) alone (P = 0.0015). Sixteen days after the second immunization with MenC-CRM(197), increased IgG (P < 0.05), IgG1 (P < 0.05), IgG2a (P = 0.06), and IgG2b (P < 0.05) were observed, and only mice primed with MenC-CRM(197) plus BCG showed affinity maturation and detectable SBA (SBA > 128). Thus, vaccination with a meningococcal conjugate vaccine (and possibly with other conjugates) may benefit from concomitant administration of BCG in the neonatal period to accelerate and enhance production of protective antibodies, compared to the current infant administration of conjugate which follows BCG vaccination at birth.

Neonatal immune response and serum bactericidal activity induced by a meningococcal conjugate vaccine is enhanced by LT-K63 and CpG2006.

Neonates have a poorly developed immune system. Therefore it is important to develop vaccination strategies that induce protective immunity and immunological memory against pathogens early in life. The immunogenicity of a meningococcal serogroup C polysaccharide conjugate (MenC-CRM(197)) was assessed in neonatal mice, and effects of LT-K63 and CpG2006 and immunisation routes were compared. Neonatal mice were primed subcutaneously (s.c.) or intranasally (i.n.) with MenC-CRM(197) with or without LT-K63 or CpG2006 and re-immunised 16 and 30 days later by the same route and formulation. Antibody levels were measured and generation of immunological memory assessed by affinity maturation and kinetics of the Ab response. Serum bactericidal activity (SBA) was measured to evaluate protective efficacy. The second and third dose of MenC-CRM(197) mixed with either LT-K63 or CpG2006 induced a rapid increase in MenC-specific IgG antibodies, to levels higher than elicited by MenC-CRM(197) alone (P<0.01) and in unimmunised mice (P<0.001), indicating efficient generation of memory by priming through both s.c. and i.n. routes. SBA was detected after three s.c. immunisations with MenC-CRM(197) s.c. alone. However, only two doses of MenC-CRM(197)+LT-K63 or MenC-CRM(197)+CpG2006 were needed to induce SBA levels>16. LT-K63 and CpG2006 enhanced neonatal antibody responses, affinity maturation, immunological memory to the conjugate MenC-CRM(197) and protective immunity. These results encourage the development of neonatal vaccination strategies to induce protective immunity and immunological memory against meningococcal disease.

Early life T cell responses to pneumococcal conjugates increase with age and determine the polysaccharide-specific antibody response and protective efficacy.

Immunization with a tetanus-protein (TT) pneumococcal polysaccharide (PPS) conjugate vaccine (Pnc1-TT) induces protective immunity against lethal pneumococcal infections in neonatal and infant mice, but anti-PPS IgG response and protective efficacy is lower than in adult mice. Here, we show that reduced antibody (Ab) response and protection against infections is directly related to impaired T cell response to the carrier. Whereas spleen cells from adult mice immunized with Pnc1-TT responded with proliferation and IFN-gamma secretion to in vitro stimulation with TT, spleen cells from neonatal and infant mice did not. However, significant, but age dependent, Th2-cytokine responses were observed in mice immunized with Pnc1-TT. Impaired IFN-gamma production upon TT-stimulation in vitro was also reflected in reduced IFN-gamma/IL-5 ratio. The IL--5 response correlated with IgG anti-PPS titers, and the lack of PPS Ab in the majority of neonatal mice was clearly associated with absence of carrier-specific IL-5 production. These results show that immunization with Pnc1-TT induces carrier-specific T cell responses that increase with age and determine the levels of PPS-specific Ab elicited. Whereas a weak and Th2-biased response was observed in neonatal mice, infant mice showed a mixed Th1-Th2 response as observed in adults.

Respiratory syncytial virus and other respiratory viruses during the first 3 months of life promote a local TH2-like response.

BACKGROUND: Respiratory syncytial virus (RSV) infections during infancy are considered to be a risk factor for developing asthma and possibly allergic sensitization. OBJECTIVE: The aim of this study was to investigate the cytokines, chemokines, and eosinophil cationic protein in the nasopharyngeal secretions of infants < or = 7 months of age with RSV infections or other respiratory viral infections and healthy infants as controls. Groups were also analyzed according to age, < or = 3 months and >3 months, and the levels were compared within and between groups. RESULTS: Thirty-nine infants with RSV, 9 with influenza or parainfluenza virus infections and 50 controls with no history of infections, were enrolled in the study. The RSV-infected infants had significantly higher levels of IL-4; macrophage inflammatory protein 1beta, a chemoattractant for T cells; and eosinophil cationic protein in nasopharyngeal secretions compared with the control group. The levels of the TH2 cytokine IL-4 were significantly higher in RSV-infected infants < or = months of age compared with RSV-infected infants >3 months of age. In infants < or = 3 months of age, infections with influenza or parainfluenza virus caused TH2-like responses similar to those produced by RSV. CONCLUSION: Infections with RSV as well as with influenza and parainfluenza virus during early infancy preferentially promote a TH2-like response in the nose with local production of IL-4, IL-5, and macrophage inflammatory protein 1beta and infiltration and activation of eosinophils.

The advantage of mucosal immunization for polysaccharide-specific memory responses in early life.

The aim of vaccination is to rapidly elicit protective immunity and generate memory for sustained protection. We studied the induction and persistence of polysaccharide (PS)-specific memory in neonatal and infant mice primed with pneumococcal conjugate (Pnc1-TT) by assessing the response to native pneumococcal PS (PPS-1), the kinetics of the PPS-1-specific IgG response to a second Pnc1-TT dose and affinity maturation. A subcutaneous (s.c.) Pnc1-TT booster induced a rapid increase in PPS-1-specific IgG, indicating efficient priming for memory by a single dose of Pnc1-TT already at 1 week of age. High levels were maintained for >12 weeks. However, a PPS-1 booster induced no response in neonatal or infant mice. The adjuvant LT-K63 significantly enhanced the IgG response and affinity to Pnc1-TT by both the s.c. and the intranasal (i.n.) route in all age groups. In neonatal and infant mice, PPS-1 and LT-K63 induced a booster response only when given i.n. following either s.c. or i.n. priming with Pnc1-TT and LT-K63. In contrast, PPS-1 with or without LT-K63 administered s.c. compromised the ongoing PPS-1-specific response elicited in neonatal mice by either s.c. or i.n. priming with Pnc1-TT and LT-K63. These results demonstrate the advantage of the mucosal route for elicitation of PS-specific memory responses in early life.

Intranasal immunization with pneumococcal conjugate vaccines with LT-K63, a nontoxic mutant of heat-Labile enterotoxin, as adjuvant rapidly induces protective immunity against lethal pneumococcal infections in neonatal mice.

Immunization with pneumococcal polysaccharides (PPS) conjugated to tetanus toxoid (TT) (Pnc-TT) elicits protective immunity in an adult murine pneumococcal infection model. To assess immunogenicity and protective immunity in early life, neonatal (1 week old) and infant (3 weeks old) mice were immunized intranasally (i.n.) or subcutaneously (s.c.) with Pnc-TT of serotype 1 (Pnc1-TT). Anti-PPS-1 and anti-TT immunoglobulin G (IgG) and IgM antibodies were measured in serum and saliva, and vaccine-induced protection was evaluated by i.n. challenge with serotype 1 pneumococci. Pnc1-TT was immunogenic in neonatal and infant mice when administered s.c. without adjuvant: a majority of the young mice were protected from bacteremia and a reduction of pneumococcal density in the lungs was observed, although antibody responses and protective efficacy remained lower than in adults. The addition of LT-K63, a nontoxic mutant of heat-labile enterotoxin, as adjuvant significantly enhanced PPS-1-specific IgG responses and protective efficacy following either s.c. or i.n. Pnc1-TT immunization. Mucosal immunization was particularly efficient in neonates, as a single i.n. dose of Pnc1-TT and LT-K63 induced significantly higher PPS-1-specific IgG responses than s.c. immunization and was sufficient to protect neonatal mice against pneumococcal infections, whereas two s.c. doses were required to induce complete protection. In addition, i.n. immunization with Pnc1-TT and LT-K63 induced a vigorous salivary IgA response. This suggests that mucosal immunization with pneumococcal conjugate vaccines and LT-K63 may be able to circumvent some of the limitations of neonatal antibody responses, which are required for protective immunity in early life.