Weaker humoral Immune Response to Tetanus Toxoid in Mice Receiving Beta-Lactam/ Ceftriaxone Treatment

2.1 Animal experiments

The experimental procedures have been validated by the Institutional Committee for the Protection and Use of Animals CEEA-14 of the University of Aix-Marseille, France and approved by the French ministry of research under file reference 201905211839232/APAFIS Number 20772. The mice were housed in specific pathogen-free conditions in group cages while respecting the number of animals per cage in the European Union EU0210 - MEPHI RITMES IHU laboratory - under approval number E1305509 after an acclimatization period of at least 7 days to avoid animal stress. Igloo-type enrichment and nesting material have been added to each cage. Daily monitoring and control of the animals was carried out by the experimenter even on public holidays. To minimize confounding factors, each cage and each mouse is processed in the same order at each stage of substance administration or blood sampling. Each cage is then returned to its original location. We used 77 BALB/cByJ male mice aged 6 to 8 weeks supplied by the Charles River Laboratory (BP 0109 L’Arbresle, 69592 Lyon, France). The health certificate for the animals complied with the specifications for the animals ordered. The experimenter, the designer, and the zoological technician were aware of the distribution of the groups at the different stages of the experiment.

Eighteen to twenty mice per group were treated with a 100 microliters intraperitoneal injection of Ceftriaxone (1g, powder for injection, VIATRIS, 1 rue de Turin, 69007 Lyon, France under reference J01DD04) at a concentration of 50 mg/kg or with 0.9% Sodium Chloride (NaCl), an isotonic saline solution for injection daily for 5 days. Ceftriaxone was prepared according to the manufacturer’s instructions at the concentration indicated above in 0.9% NaCl (From AGUETTANT, Quai Louis Aulagne, 69190 Saint-Fons, France, Code ATC B05XA03) and based on results described by Majhi et al.²⁰

Mice received a single dose of DTP (diphtheria-tetanus-polio) vaccine purchased from the Sanofi-Winthrop manufacturer (product REVAXIS, ATC code J07CA01, Sanofi Winthrop Industrie, 83 avenue Raspail, 94250 Chantilly, France; batches R3J398V, R3J391V, T3C215V). Two different vaccine administration protocols (Conditions A or B, see below) were studied and for each condition, mice were divided into groups receiving either the antibiotics ceftriaxone or a solution of isotonic saline (0.9% NaCl) as a control. Condition A: immunization before the start of antibiotics (or control) treatment (Groups 1C & 1D/ 20 mice/ Ceftriaxone and Groups 1A & 1B/ 19 mice/ 0.9% NaCl – controls), and Condition B: immunization after the end of antibiotics (or control) treatment (Groups 2G & 2H/20 mice/Ceftriaxone and Groups 2E & 2F/18 mice/0.9% NaCl – controls), as illustrated in Figure 2. Each group (under each condition) was compared: 1- to other groups in the same condition, 2-to groups in the other condition and 3- to placebo (NaCl) control groups. We excluded older mice to retain only the groups of animals exhibiting a strong immune response. We considered all animals from all groups (insufficient sample size for testing). The sole reason for the reduction in sample size (n) stems from the number of samples where the quantities were insufficient, as shown in particular by the decrease in n (number of mice) in Figures 3 and 4.

Figure 2. Schema: DTP immunization before or after Antibiotic administration (Ceftriaxone) or non-antibiotic administration (NaCl, as control).

20 male BALB/cByJ mice were immunized by intraperitoneal injection of DTP Vaccine four days before antibiotic treatment during 5 days (Red/ 1-A/Condition A). 20 male BALB/cByJ mice were immunized by intraperitoneal injection of DTP Vaccine four days after beginning of antibiotic (Green/1-B Condition B) treatment during 5 days. Blood from mice was collected at Day-5 for preimmune control, and at Day8, D14, D21and D42, final blood collection.

Figure 3. Graphs: Kinetics of immune responses of mice immunized with DTP vaccine under non-antibiotic administration (NaCl).

A- DTP Immunization before 0.9% NaCl treatment (As Condition A), B- DTP Immunization after 0.9% NaCl treatment (As Condition B). Between 5 to 7 serum mice per day on each treated group was quantified as described in M&M. The median of each mice group by days (D8, D14, D21 and D42) was calculated and plot of each serum value is represented. *** p value= 1.03e-05.

Figure 4. Graphic: anti-tetanus immune response quantification at D42 of vaccinated mice under Ceftriaxone treatment.

Mice were vaccinated with DTP vaccine before or after Ceftriaxone (CFTX) treatment (n = 10) versus 0.9%NaCl as “control”/NaCl (n = 9 and 6). An Anova test give us a significant effect between both treatment (p = 1.31e-10). Condition A or B gave us also a significant p value (p = 0.00167).

We selected the commercial three-component DTP vaccine because it is a vaccine commonly used in humans and tested on mice before obtaining marketing authorization.²¹ The dose of vaccine used was 150 microliters by intraperitoneal injection (with a 25G needle of 16 mm length), i.e. a concentration of 6 IU of TT/mouse. This dose was calculated based on data from Huh DH, et al. and the dose tests carried out in our laboratory to have a sufficiently detectable signal with the Immunochromatographic test we chose after a single injection of antigens (Data not shown).²² The active substances in each 0.5 ml dose of vaccine are: - Purified diphtheria toxoid not less than 2 IU* - Purified tetanus toxoid (TT) not less than 20 IU* - Inactivated poliomyelitis virus (produced in Vero cells) --Type 1: 40 antigen D units** --Type 2: 8 antigen D units** --Type 3: 32 antigen D units** * IU is an international unit for measuring vaccine potency ** the antigen unit expresses the amount of antigen present in the vaccine dose.

Intermediate and pre-immune blood samples were taken using a submandibular lancet. A final blood sample was taken intracardially after anesthesia and euthanasia of each mouse. Each blood sample was centrifuged at 800 rpm at 4°C during 15 minutes and sera samples were then stored at -30°C until used for analysis.

As described in the May 2019 Project Authorization Application (PAA), general anesthesia is achieved by intraperitoneal (IP) injection of a mixture of ketamine (90 mg/kg) and xylazine (10 mg/kg) in mice. Ketamine was marketed under the name Imalgene 1000 by VIRBAC (13 rue LID, 05617 Carros, France) and xylazine under the name ROMPUN 2% by BAYER HealthCare SAS - Animal Health (1 rue Claude Bernard, 59000 Lille, France). The mice were then euthanized by IP injection of pentobarbital sodium (CEVA Santé Animale, 8 rue de Logroño, 33500 Libourne, France) or DOLETHAL containing pentobarbital (100 mg/kg) (VETOQUINOL, 10 rue du Docteur Deubel, 70200 Lure).

2.2 Tetanus serum analysis

We particularly followed the anti-tetanus response because it was demonstrated that in male mice of different age (the youngest and the oldest mice only), the cellular and humoral immune responses are not altered unlike those against diphtheria toxin.²³ Tetanus antitoxin sera were analyzed by Immunochromatographic test with the “TQS TETANOS QUICK STICK” kit, referenced # TE-20B/S-GZ-20B from Eurobio Scientific (7 av. De Scandinavie, ZA de Courtaboeuf, 91940 Les Ulis, France). Following the manufacturer’s procedure, twenty microliters of each serum were dispensed in the well of the stick and three drops of the diluent provided by the kit was immediately added to the sample. The results are read after no ten minutes by scanning the stick using the “Image Scanner 3” (GE Healthcare, Buc, Yvelines, France). Scanned sticks were formatted for alignment by mouse batch and treatment group using Adobe Photoshop CS3 software. Quantification of each stick was calculated by measuring band intensities using the “Image Quant TL Version 7.0 analysis software (GE Healthcare, Buc, Yvelines, France). In Figures 2 and 3, the values on the y axis represent the relative intensity of the band (area) of the pink T Test line quantified by the software.

2.3 Statistics

The statistical analysis and Figures 3 and 4 result from the free R software used for statistical calculation and graphics. An Anova test was used to calculate p values for the analysis in Figure 3 to find out if there is a significant difference between the two conditions (before or after isotonic saline treatment) and a significance difference of time for the two conditions. For Figure 4, an Anova test gave a significant result for the condition effect and time, so a non-parametric Wilcoxon test was used to calculate p-values to confirm the significant effect.

3.1 Evaluation of the immunosuppressive role of ceftriaxone in mouse

Our goal was to evaluate whether Ceftriaxone alters DTP humoral immune response.

For this purpose, we decided to use an animal model to experimentally follow the antibody response against Tetanus Toxin in groups of DTP immunized mice before (CONDITION A), after (CONDITION B) antibiotics treatment or not submitted to any antibiotic treatment as a control group as shown in Figure 2.

Thus, we chose to examine the Tetanus Toxin immune response after DTP vaccine administration in mice treated with Ceftriaxone (n = 40) versus those treated with isotonic saline solution as control (n = 37). A group of mice was immunized before the start of Ceftriaxone treatment or with isotonic saline solution (n = 20/19, respectively). ( Figure 2A), and two groups were immunized after treatment with Ceftriaxone or isotonic saline solution (n = 20/18, respectively) ( Figure 2B). We collected the pre-immune blood samples five days before the start of the Ceftriaxone or saline treatment (Day-5) for each mouse. During the experiment, we then took blood from the mice at Day8, D14, D21 and D42 (the final blood collection).

We monitored the anti-tetanus response with the Tetanus Quick Sticks kit used in the context of a vaccination schedule or a suspicion of Clostridium tetani infection diagnosis in our laboratory. The advantage of this technique is that it is easy to use, consumes very little serum (or whole blood, 20 microliters) and that results are obtained quickly (less than 10 minutes). This kit is based on immunochromatography assay principle. A mixture of gold conjugates coupled with tetanus toxoid and BSA is fixed on the solid phase of the stick. The mouse serum sample to be tested is placed in the well of the stick, followed by a few drops of diluent. The migration of the diluent onto the stick carries the gold conjugate which forms a complex with the anti-tetanus immunoglobulins present in the sample. If samples are positives, complexes between gold conjugates and anti-tetanus immunoglobulins, stopped on the immobilized toxoid on the stick and appear in the stick window in lane T. The control band, C pink lane in the window, is formed by the capture of the excess of gold conjugates by the control reagent constituted of poly-L-Lysine based buffer ( Figure S1).

3.2 Dose-response adjustment post-DTP immunization

Before attempting to answer the question about the potential alteration of the immune response by Ceftriaxone treatment, we tested the efficacy of the immunological responses in mice immunized with the human DTP vaccine. The response dose was established at 6IU per mouse as described in M&M based on D.H. Huh et al. data.²² By immunizing the mice with the DTP vaccine at 6IU, we were able to detect anti-Tetanus Toxin immune response with the specific kit we chose: the object was not to protect them against tetanus but to measure whether they produced antibodies against an antigen under β-lactams family’s antibiotic treatment or not (Data not shown). Then, we followed the kinetics of the immune responses of immunized mice with the DTP vaccine before ( Figure 2A) and after ( Figure 2B) the isotonic saline treatment. This control treatment consisted of a five-day daily intraperitoneal injection of a 0.9% sodium NaCl solution. We observed in scanned sticks ( Figure S1) that, for the eight randomly chosen mice (four for each condition), no band appeared in the pre-immune serum P0 samples in the Test T line compared to the control C line which guaranteed that the analyzed sticks were valid. Then, from day 8 to day 42, we observed a band corresponding to the detection of anti-tetanus toxin antibodies in the serum of the majority of the mice. Notably, the intensity of signal gradually increased over time, particularly from day 14 to day 42 in the test line.

The Figure 3 presents the relative intensity of quantified bands of each stick from immunized mice serum in the two tested condition (A and B). We observed that, as generally observed in literature, the maximum immune response after a natural exposure to antigens or to one dose of vaccine, occurred around 4-6 weeks after injection, as we can see for D42 in our Figure 3.²⁴ There was a significant difference in the immune response over time (from D8 to D42, last day test) similar for the two conditions A and B (p value= 1.03e-05).

We also observed no significant difference in TT-response in both conditions: before (Condition A) or after (condition B) isotonic saline treatment from Day 8 to Day 42.

The date-to-date variability [antibodies were not measured daily] in anti-Tetanus Toxin antibody responses per mouse could be explained by animal-to-animal variability.

Despite the high value at D8 in condition B for two mice, the mean values remained very close to condition A. At Day 8, in condition B, the discrepancy between the minimum and maximum values and the high anti- Tetanus Toxin antibody responses can both be explained by the fact that, in this particular condition, the DTP injection took place just 5 days before the D8 blood collection. Conversely, in condition A, the injection of DTP was carried out twelve days before, which may explain a weaker anti-Tetanus Toxin antibody response.

By observing all the results obtained in Figures 3 and Figure S1, we decided to compare in the rest of the study, the anti-tetanus serological responses at D42 only, where the quantification signal was the highest.

3.3 Ceftriaxone inhibits the humoral immune response

Figure 4 summarizes all immune responses against tetanus under the two conditions in each group of mice for both treatments at D42. An Anova test give us a significant effect between both treatment (p = 1.31e-10). Condition A or B gave us also a significant p value (p = 0.00167). We then calculated p-values with a non-parametric statistic test and we observed a significant drop of 35% in terms of p-values between the mean values of the anti-tetanus immunoglobulins in the sera of the mice immunized after treatment with Ceftriaxone compared with those measured in the mice immunized before the same treatment (p value = 0.02881 for Wilcoxon test). The effect “before or after treatment” during isotonic saline was much less marked than during the Ceftriaxone treatment. In isotonic saline treatment there was no significant difference between the two conditions (p value = 0,5949 for Wilcoxon test).