Infect Immun. 2015 Aug;83(8):3157-63. doi: 10.1128/IAI.00258-15.

Four-component Staphylococcus aureus vaccine 4C-staph enhances Fcγ receptor expression in neutrophils and monocytes and mitigates S. aureus infection in neutropenic mice

Antonina Torre,a Marta Bacconi,a Chiara Sammicheli,a Bruno Galletti,a Donatello Laera,a Maria Rita Fontana,a Guido Grandi,a* Ennio De Gregorio,a Fabio Bagnoli,a Sandra Nuti,a Sylvie Bertholeta and Giuliano Bensia#

Novartis Vaccines and Diagnostics srl, a GSK Company, Via Fiorentina 1, 53100 Siena, Italya

# Address correspondence to Giuliano Bensi,

*Present address: Guido Grandi, Center for Integrative Biology, University of Trento, Italy



Staphylococcus aureus is a human bacterial pathogen causing a variety of diseases. The occurrence of multidrug resistant strains of Staphylococcus aureus underlines the need for a vaccine. Defining immune correlates of protection may support the design of an effective vaccine.

We used a murine Staphylococcus aureus infection model, in which bacteria were inoculated in an air-pouch generated on the back of the animal. Analysis of the air-pouch content in mice immunized or not with an adjuvanted multiantigen vaccine formulation (4C-Staph) prior to infection allowed us to measure bacteria, cytokines and 4C-Staph-specific antibodies, and to analyze host immune cells recruited to the infection site.

Immunization with 4C-Staph resulted in accumulation of antigen-specific antibodies in the pouch and mitigated the infection. Neutrophils were the most abundant cells in the pouch, and they showed up-regulation of Fcγ III/II receptor (FcγR) following immunization with 4C-Staph. Reduction of the infection was also obtained in mice immunized with 4C-Staph and depleted of neutrophils, which showed an increase in monocytes and macrophages.

Up-regulation of the FcγR and presence of antigen-specific antibodies induced by immunization with 4C-Staph may contribute to increase bacterial opsonophagocytosis. Protection in neutropenic mice indicated that an effective vaccine could activate alternative protection mechanisms compensating neutropenia, a condition often occurring in Staphylococcus aureus infected patients.

PMID: 26015481



Staphylococcus aureus (S. aureus) is a human bacterial commensal which can occasionally turn into an opportunistic pathogen, causing a variety of pathologies. The greatest burden of morbidity is due to skin and soft tissue infections, which can be either uncomplicated or can spread to deeper tissues and require hospitalization and sometimes surgery (1). The current emergence of strains which are resistant to multiple antibiotics (2), makes the treatment of S. aureus infections more difficult, underlining the medical need for a S. aureus vaccine, which is not yet available.

Increasing our knowledge on S. aureus-induced pathogenesis and identifying immunological correlates of protection are key steps towards the development of an effective vaccine (3, 4). We recently described an aluminum hydroxide-adjuvanted vaccine formulation, 4C-Staph, which was able to protect mice from infection in different murine models (5). The use of systemic infections hampered a deeper analysis of immune humoral factors and cellular components possibly associated with protection at the site of infection. Therefore, this prompted us to use an infection model that would allow the concomitant evaluation in situ of different parameters related to both infection and host response.

The model is based on two dorsolateral subcutaneous injections of air to generate a “pouch” in which bacteria are subsequently inoculated, mimicking a skin infection. Then, the content of the pouch can be retrieved allowing the evaluation of multiple parameters such as number of bacteria, recruitment of host live immune cells, and presence of antigen-specific antibodies and cytokines release.


Figure 1

FIG 1  4C-Staph-immunized mice show antigen-specific antibodies in the pouch and mitigate the infection and the inflammation in situ. A, CFU measured in the pouch lavage 48 h post-infection. CFU values obtained for single mice are represented by black dots, while grey lines indicate the median values. Statistical significance was assessed with the two-tailed Mann Whitney test and the P value is indicated. B and C, Bar graphs reporting the antibody responses elicited against each antigen of the 4C-Staph and measured in the sera (B) and air pouch lavages (C) of animals immunized with either alum (grey bars) or 4C-Staph (black bars). Bars indicate the geometric mean of the fluorescence intensities (MFI) with 95 % confidence interval (C.I.) obtained from four independent experiments, for a total of 63 mice for sera and one representative experiment with 6 mice for pouch lavages. Groups immunized with alum highlight the assay background threshold for each antigen. D, Bar graph showing cytokine levels measured 48 h post-infection in the pouch lavages of mice immunized with either alum or 4C-Staph and then either mock-infected or infected with S. aureus Newman. Bars indicate the geometric mean (with 95 % C.I.) of four S. aureus infection experiments (19-23 mice per group) and one representative experiment for mock-infected mice (6 mice per group). The statistical analysis was performed using the Mann Whitney two-tailed test (*P<0.05, **P<0.01).


After having established that vaccination of mice resulted in reduction of CFUs in the air pouch (Fig. 1A), we further investigated the host immune responses. Sera and pouch lavages were collected from immunized mice to measure 4C-Staph-specific antibodies and secreted cytokines. Fig. 1 shows that immunization of mice prior to infection elicited detectable antigen-specific antibodies that were measured in the serum (Fig. 1B) and in the air pouch (Fig. 1C). This finding prompted us to assess the protective role of the humoral response in the air pouch model as well. Passive administration of 4C-Staph-specific antiserum significantly reduced the number of CFU in the pouch lavage (Fig. 2A), confirming that antibodies play a role in protecting mice from S. aureus infection in situ.

Cytokines, as well as host immune cells recruited in the pouch, were also evaluated in immunized and infected mice 48 h post-infection. Immunization with 4C-Staph significantly reduced the in situ levels of some of these cytokines compared to alum alone, indicating that vaccination with 4C-Staph reduces not only the bacterial burden, but also the inflammation in situ (Fig. 1C). Recruitment in the pouch of host immune cells was also investigated.

Using the cytofluorimetric analysis, multiple cell populations were identified, in particular neutrophils, eosinophils, macrophages, dendritic cells (DC), natural killer (NK), B and T cells. Forty-eight hours following infection, the total number of live cells significantly increased in the pouch, with neutrophils, monocytes and macrophages showing the sharpest increase (Fig. 2 B, C, D), in agreement with the role that these cell types have in the initial phases of the host response against the infection. Neutrophils were the most abundant cell population (around 90 %), followed by monocytes, macrophages and eosinophils. No significant differences were observed between mice immunized with 4C-Staph and those immunized with alum in the number of the cells recruited to the infection site (Fig 2. B, C, D)


Figura 2

FIG 2   4C-Staph elicits functional antibodies. Host recruited cells in the pouch of infected mice. A, CFU measured in the pouch lavages of mice passively immunized with rabbit antisera raised against either alum or 4C-Staph. CFU counts in individual animals are represented by black dots (three independent experiments, 31 mice). Statistically analyses were performed using the two-tailed Mann Whitney test. B, C and D Bar graphs reporting the absolute cell numbers of neutrophils (B), monocytes (C) and macrophages (D) in mice immunized either with alum (grey bars) or with 4C-Staph (black bars) and then either mock-infected or infected with Newman strain. Bars represent the geometric mean with a 95 % C.I. from 2-5 independent experiments (12-30 mice total/per group).


We therefore investigated whether immunization with 4C-Staph might induce qualitative changes in the recruited immune cell populations. Levels of expression of activation markers like Fcg receptor (FcgR) III/II (CD16/32) were analyzed on the surface of monocytes, macrophages and neutrophils, the three most abundant cell populations in the pouch. Neutrophils and monocytes recovered from the pouch of immunized and infected mice showed an up-regulation of the FcgR III/II molecules (Fig. 3). FcgR III/II and other FcgRs, are key receptors on human and murine neutrophils, monocytes, DC, and macrophages, which recognize antibody-coated bacteria and infected cells, and favor their elimination by phagocytosis and antibody-dependent cellular cytotoxicity (6, 7). The upregulation of the FcγR III/II on neutrophils and monocytes suggests a transition of the cells from the resting to an activated state. At this stage, neutrophils, armed with functionally activated FcγRIII/II complexes, would more efficiently take up the bacteria coated with antigen-specific antibodies.


Figura 3

FIG 3   4C-Staph increases the expression of FcgR III/II on neutrophils and monocytes. Graphs showing the FACS fluorescent intensity signals (FI) detected with an anti-CD16/32 antibody directed against the FcgR III/II receptors on neutrophils (A) and monocytes (B) in the pouch lavages of mice immunized either with alum (empty triangles) or with 4C-Staph (black dots) and then either mock-infected or infected with Newman strain. Grey lines indicate the median among independent experiments. Statistical analyses were performed using the Mann Whitney two-tailed test.


With the goal of confirming the major role played by neutrophils in containing the infection, we immunized mice either with 4C-Staph or with alum, followed by neutrophil depletion and infection with S. aureus. Surprisingly, a significant reduction (P = 0.002) in the CFU count was observed in the pouch lavages of neutropenic animals immunized with 4C-Staph, as compared with the control group of neutropenic mice vaccinated with alum (Fig. 4A). We hypothesized that in absence of neutrophils the immunization with 4C-Staph may have activated compensatory protection mechanism(s) that mediated the control of the infection. The analysis of the cells recruited in the pouch revealed that immunization with 4C-Staph resulted in significant increases of the monocytes and macrophages populations in the neutropenic mice (Fig. 4B and C).

These results suggested that monocytes and macrophages might compensate for neutrophil deficiency and mediate 4C-Staph-dependent protection.

These findings may have important implications for vaccine development since neutropenia in humans is one of the pathological conditions that make patients most vulnerable to S. aureus infection.


Figura 4

FIG 4  Immunization of neutrophil-depleted mice with 4C-Staph mitigates the infection in situ and increases macrophages and monocytes recruitment. A, CFU at 48 h post infection in the pouch of anti-Ly6G-treated mice immunized either with alum or with 4C-Staph. Grey lines indicate the median from 4 independent experiments. B and C, ratios between the absolute cell numbers of macrophages (B) and monocytes (C) of each neutropenic mouse and the median of the absolute cell numbers of the same populations recovered in the immunocompetent corresponding groups. Ratios obtained for individual neutropenic mice are reported for groups immunized either with alum or with 4C-Staph. Grey lines indicate the median values of the ratios for each group (three independent experiments). Statistical significance was calculated using the two-tailed Mann Whitney test.


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