A randomized, controlled, blinded study of the safety and immunogenicity of Haemophilus influenzae type b conjugate vaccine injected at different intramuscular sites in Chinese infants

Abstract

To compare the safety and immunogenicity of Haemophilus influenzae type b (Hib) conjugate vaccine administered via the vastus lateralis and deltoid muscles, 320 healthy Chinese infants <12 mo of age were enrolled in a randomized, controlled, blinded study and divided into 2 age groups: 2–5 mo and 6–12 mo. Each age group was then randomized (1:1) to either the vastus lateralis (experimental) group who received Hib vaccination into this muscle 2 or 3 times at monthly intervals, or the deltoid (control) group who received Hib vaccination into this muscle either 3 times (2–5 mo group) or twice (6–12 mo group) at monthly intervals. Local and systemic adverse reactions after each vaccine dose were recorded, and Hib-PRP antibody concentrations were determined by ELISA at 28 d after completion of the immunization schedule. There were no significant differences in the proportions of subjects with post-immunization Hib-PRP antibody concentrations ≥1.0 μg/mL or ≥0.15 μg/mL with the two injection sites for either age group, or in the post-immunization Hib-PRP antibody concentrations achieved (P > 0.05). In addition, there were no significant differences in the rates of local and systemic reactions after the first and second vaccinations between the 2 injection sites for either age group (P > 0.05), but the rate of systemic reactions in the 2–5 mo group after the third vaccination via the vastus lateralis muscle was significantly lower than after deltoid vaccination (0% vs 8.57%; P < 0.05). Thus, administration via the vastus lateralis muscle is worth considering for Hib vaccination.

Keywords: :

• Haemophilus influenzae type b vaccine
• vaccination site
• vastus lateralis
• deltoid
• safety
• immunogenicity

Introduction

Vaccination is the most effective means of preventing infectious diseases and, as an important part of public health services, has social significance. Currently, most countries around the world have included vaccinations in their national health strategies. With an increasing understanding of disease pathogenesis and the ongoing development and application of newer vaccines, more and more opportunities to prevent the occurrence of infectious diseases have become available to clinicians.

The gluteus maximus and deltoid muscles have long been the most commonly employed vaccination sites. However, injection into the buttocks can result in sciatic nerve damage, especially in infants, and since it requires the body to be exposed, this route is inconvenient for patients and is no longer commonly used.¹ As the upper and central parts of the deltoid muscle are easy to locate and there are no major nerves and blood vessels present, this site has gained in popularity for vaccination.² In infants, however, there is less upper arm muscle distribution, and multiple injections into the deltoid may result in induration.¹ Therefore, it is imperative to select an accessible and safe alternative vaccination site, especially in view of the numbers of vaccines that are given nowadays.

When choosing an injection site, safety should be the first consideration and convenience should be the second, especially in infants and young children. The vastus lasteralis muscle has no large blood vessels and nerves and has sufficient thickness to make it easy to grasp, which makes it an ideal site for intramuscular injections in infants.¹ Studies have shown that vastus lateralis muscle injection of commonly used therapeutic drugs such as penicillin and vitamins is associated with a low incidence of nerve injury, local infection and induration.^3-5 However, there are limited studies of vastus lateralis injection for vaccination, and therefore its application for this purpose and its impact on vaccine safety and immunogenicity is worth studying. The only studies of Haemophilus influenzae type b (Hib) vaccine administered via the vastus lateralis muscle have reported an incidence of about 5% for systemic and local adverse reactions, which was not significantly different to that with administration via deltoid and gluteal muscles.^1,6 In the study of Junqueira et al.⁷ reported in 2010, there was no statistically significant difference between hepatitis B vaccination administered via the gluteal and vastus lateralis muscles, with antibody conversion rates of 97.8% and 97.6%, respectively.

As there have been no reports of the safety and immunogenicity of Hib vaccination via the vastus lateralis muscle, we performed a randomized, controlled, blinded study in 320 healthy infants less than 12 mo of age to compare the safety and immunogenicity of Hib vaccination administered via the vastus lateralis and deltoid muscles.

Results

Subjects and baseline analysis

A total of 320 infants met the inclusion/exclusion criteria for the study and were randomized to the experimental (vastus lateralis) and control (deltoid) study groups (160 in each in group). During the study, 22 infants dropped out (in 6 cases because their guardians did not agree to attend follow-ups while 16 infants were lost to follow-up); 298 (93.13%) infants completed the safety observations (152 in the experimental group and 146 in control group), and 290 (90.62%) had serum antibody level data available from paired (pre- and post-immunization) blood samples (Table 1). There were no statistically significant differences in subject ages between the experimental and control groups (p > 0.05; Table 1), nor in the pre-immunization Hib-PRP antibody geometric mean concentrations (GMCs) between the experimental and control groups (p > 0.05; Table 2).

Table 1. Numbers and mean ages of infants in the study groups

Age group	Injection site	No. of subjects	Mean age ± SD (months)	No. completing observations	No. with paired serum samples
2–5 mo	Deltoid	80	2.74 ± 1.05*	70	70
	Vastus lateralis	80	2.58 ± 0.92	73	73
6–12 mo	Deltoid	80	8.48 ± 1.42^†	76	71
	Vastus lateralis	80	8.30 ± 1.58	79	76

* p = 0.490 vs vastus lateralis group. ^†p = 0.471 vs vastus lateralis group.

Table 2. GMCs of Hib-PRP antibodies before immunization in the vastus lateralis (experimental) and deltoid (control) groups

Age group	Injection site	No. of subjects	Antibody GMCs (μg/mL)	P value
2–5 mo	Deltoid	70	0.36 ± 0.48	0.374
	Vastus lateralis	73	0.37 ± 0.50
6–12 mo	Deltoid	71	0.87 ± 0.91	0.648
	Vastus lateralis	76	0.67 ± 1.34

GMC, geometric mean concentration; Hib, Haemophilus influenzae type b; PRP, polyribosylribitol phosphate.

Immunogenicity results

The proportions of subjects with Hib-PRP antibody GMCs ≥ 1.0 μg/mL in the 2–5 mo age group were 95.71% and 95.89% with administration via the deltoid and vastus lateralis muscles, respectively (p > 0.05); these percentages rose to 97.18% and 97.37%, respectively, in the 6–12 mo age group (p > 0.05). The proportions with Hib-PRP antibody GMCs ≥ 0.15 μg/mL reached 100% in both age groups with both deltoid and vastus lateralis administration (Table 3).

Table 3. Antibody protection rates after immunization in the experimental (vastus lateralis) and control (deltoid) groups

Age group	Injection site	No. of subjects	Hib-PRP antibody GMC			Hib-PRP antibody GMC
			≥ 1.0 μg/mL			≥ 0.15 μg/mL
			No.	Percentage	P value	No.	Percentage	P value
2–5 mo	Deltoid	70	67	95.71	1	70	100	‒
	Vastus lateralis	73	70	95.89		73	100
6–12 mo	Deltoid	71	69	97.18	1	71	100	‒
	Vastus lateralis	76	74	97.37		76	100

GMC, geometric mean concentration; Hib, Haemophilus influenzae type b; PRP, polyribosylribitol phosphate.

There were also no significant differences in the Hib-PRP antibody levels achieved post-immunization after administration via the 2 injection sites. Hib-PRP antibody GMCs after deltoid and vastus lateralis muscle administration were 5.13 ± 42.95 μg/mL and 5.33 ± 43.76 μg/mL, respectively, in the 2–5 mo age group (p > 0.05), and 2.49 ± 29.57 μg/mL and 3.07 ± 33.31 μg/mL, respectively, in the 6–12 mo age group (p > 0.05; Table 4).

Table 4. GMCs of Hib-PRP antibodies after immunization of the experimental (vastus lateralis) and control (deltoid) groups

Age group	Injection site	No. of subjects	Antibody GMCs (μg/mL)	P value
2–5 mo	Deltoid	70	5.13 ± 42.95	0.755
	Vastus lateralis	73	5.33 ± 43.76
6–12 mo	Deltoid	71	2.49 ± 29.57	0.276
	Vastus lateralis	76	3.07 ± 33.31

GMC, geometric mean concentration; Hib, Haemophilus influenzae type b; PRP, polyribosylribitol phosphate.

Safety findings

Local adverse reactions occurring after vaccination mainly included induration and swelling, which were mild and had a short duration of up to 3 d. A total of 19 infants (6.38%) exhibited local reactions after the first vaccination dose, but none did so after the second dose, and only 1 (0.34%) had a local reaction after the third dose (Table 5). There were no statistically significant differences in local reaction rates after each vaccination dose between the deltoid and vastus lateralis injection sites (p > 0.05).

Table 5. Local reactions during 28 d after each vaccination in the experimental (vastus lateralis) and control (deltoid) groups

Age group	Injection site	No. of subjects	No. with local reactions			Percentage (total)	χ^2	P value
			Induration	Redness	Swelling
• Following the first dose:
2–5 mo	Deltoid	70	1	6	1	11.43	0.805	0.370
	Vastus lateralis	73	0	9	3	16.67
6–12 mo	Deltoid	76	0	0	0	0	‒	‒
	Vastus lateralis	79	0	0	0	0
• Following the second dose:
2–5 mo	Deltoid	70	0	0	0	0	‒	‒
	Vastus lateralis	73	0	0	0	0
6–12 mo	Deltoid	76	0	0	0	0	‒	‒
	Vastus lateralis	79	0	0	0	0
• Following the third dose:
2–5 mo	Deltoid	70	0	1	0	1.43	1.036	0.309
	Vastus lateralis	73	0	0	0	0

Systemic adverse reactions after vaccination mainly included fever, rash, vomiting, abnormal crying, somnolence, loss of appetite, and irritability. Among these, abnormal crying and irritability had the highest incidences and occurred mainly after the first vaccination dose in the 2–5 mo age group (Table 6). Individual subjects also exhibited diarrhea symptoms. However, all systemic reactions were mild and of short duration, causing no harm to any of the infants. A total of 78 infants (26.17%) exhibited systemic reactions after the first vaccination dose, as compared with 5 (1.68%) after the second dose, and 6 (2.01%) after the third dose. Although there were no statistically significant differences in the incidences of systemic reactions between deltoid and vastus lateralis muscle injection sites after the first and second vaccination doses (p > 0.05), the incidence of systemic reactions with vastus lateralis administration was significantly lower than with deltoid administration after the third vaccination dose in the 2–5 mo age group (0% vs 8.57% respectively; p < 0.05) [Table 6].

Table 6. Systemic reactions during 28 d after each vaccination in the experimental (vastus lateralis) and control (deltoid) groups

Age group	Injection site	No. of subjects	No. with systemic reactions								Percentage (total)	χ^2	P value
			Fever	Rash	Vomiting	Abnormal crying	Somnol- ence	Loss of appetite	Irritab- ility	Other
• Following the first dose:
2–5 mo	Deltoid	70	11	2	1	9	3	4	11	1	60.00	1.434	0.231
	Vastus lateralis	73	4	0	0	17	0	3	12	0	50.00
6–12 mo	Deltoid	76	0	0	0	0	0	0	0	0	0	‒	‒
	Vastus lateralis	79	0	0	0	0	0	0	0	0	0
• Following the second dose:
2–5 mo	Deltoid	70	0	0	0	0	0	1	0	0	1.43	1.779	0.182
	Vastus lateralis	73	0	0	0	1	0	1	2	0	5.56
6–12 mo	Deltoid	76	0	0	0	0	0	0	0	0	0	‒	‒
	Vastus lateralis	79	0	0	0	0	0	0	0	0	0
• Following the third dose:
2–5 mo	Deltoid	70	2	1	0	1	0	1	1	0	8.57	5.924	0.015
	Vastus lateralis	73	0	0	0	0	0	0	0	0	0

Discussion

The vaccination site and vaccine dose are keys to successful vaccination.¹ Choosing the best inoculation site is important for ensuring safe and effective vaccination, particularly for infants. Over the years, gluteus maximus and deltoid muscles have been the most commonly used vaccination sites. However, as studies have shown that hip muscles are generally not fully developed in infants less than 2 y of age, especially in neonates, use of the gluteus maximus for intramuscular injection is not appropriate as it may result in sciatic nerve damage. In the case of the deltoid, there is less upper arm muscle distribution in infants, and multiple injections at this site can result in induration. With the expansion of immunization programs for children, increasing types of vaccination and inoculation times, continued use of the deltoid muscle may become difficult. Thus, selecting an injection site to ensure optimal vaccination of infants is a challenge.

The present study was designed to compare the safety and immunogenicity of Hib vaccination administered via the vastus lateralis and deltoid muscles, and thus provide a preliminary basis to select a safe and effective alternative immunization site. The study showed that the proportions of infants with post-vaccination Hib-PRP antibody concentrations ≥ 1.0 μg/mL in the 2–5 mo age group were 95.71% and 95.89% after deltoid and vastus lateralis administration, respectively, and 97.18% and 97.37%, respectively, in the 6–12 mo age group. Consequently, there was no statistically significant difference in the proportions of infants achieving Hib-PRP antibody concentrations adequate for long-term protection between the 2 injection sites (p > 0.05). Hib-PRP antibody concentrations ≥ 0.15 μg/mL were achieved in 100% of subjects with vaccination at each injection site. Thus, there were no statistically significant difference in Hib-PRP antibody concentrations appropriate for both long-term protection and natural protection between deltoid and vastus lateralis vaccination, indicating that vastus lateralis inoculation does not influence the immunogenicity of the vaccine.

In addition, safety findings showed that there were no statistically significant differences in the incidences of local adverse reactions after each vaccination dose when the Hib vaccine was administered via the deltoid and vastus lateralis muscles (p > 0.05). While there were also no statistically significant differences in the incidences of systemic reactions after the first and second vaccine doses with deltoid and vastus lateralis administration (p > 0.05), the incidence of systemic reactions after the third vaccination dose in the 2–5 mo age group with vastus lateralis muscle injection was significantly lower than with deltoid muscle injection (p < 0.05).

Although the incidence of adverse events with Hib vaccination in our study was slightly higher than that reported in other studies conducted in China,^1,6 it is worth noting that the main adverse events observed in our study were systemic reactions such as abnormal crying and irritability, whereas the main adverse events reported in other studies have been other reactions such as fever.^8-10A total of 26 cases of abnormal crying occurred in infants 2–5 mo of age after the first vaccination in our study (18.18% of this age group) and there were 23 cases of irritability (16.08%), but there were only 15 cases of fever (10.49%). In comparison with deltoid muscle injection, vastus lateralis injection did not increase the incidence of either systemic or local adverse reactions in this study, and this finding together with the potential advantage of giving multiple injections into the vastus lateralis muscle rather than into the deltoid muscle suggest its usefulness in infants 2–5 mo of age.

In conclusion, the results of this study indicate that vastus lateralis muscle injection did not affect the immunogenicity and safety of Hib vaccination in infants less than 12 mo of age, and in younger infants (2–5 mo of age) was associated with fewer systemic adverse reactions after the third dose than injection into the deltoid muscle. Thus, vastus lateralis injection is worthy of consideration for Hib vaccination of infants.

Materials and Methods

Study design and subjects

We performed a randomized, controlled, blinded study in healthy infants aged 2 to 12 mo without autoimmune diseases who were not currently suffering from fever or acute illnesses, had no history of allergy or Hib-related diseases, and had not previously received Hib vaccination. The infants were divided into two age groups in a 1:1 ratio ‒ one group was 2–5 mo of age and the other 6–12 mo of age. Each age group was then randomized (1:1) to an experimental (vastus lateralis muscle inoculation) group and a control (deltoid muscle inoculation) group via SAS 8.1 (20 per block; 10 to vastus lateralis injection and 10 to deltoid muscle injection). Prior to vaccination, neither the investigator nor the patients knew which site would be used for injection, and when observing for local and systemic adverse reactions, the investigator did not know which site had been used. The immunization schedule was as follows: Infants in the experimental group received the Hib vaccine 2 or 3 times into the vastus lateralis muscle 1 mo apart. In the control group, the Hib vaccine was administered into the deltoid muscle 3 times in infants 2–5 mo of age and twice in infants 6–12 mo of age, each dose being given a month apart. All immunizations completed before the validity date of the study Hib vaccine, that is 30 April, 2012.

Ethical Committee approval for the study was obtained, and it was conducted in accordance with the provisions of the Declaration of Helsinki. Participation in the study was voluntary. Informed consent was obtained from parents/guardians prior to its commencement.

Vaccine

The Haemophilus influenzae type b conjugate vaccine used in the study was manufactured by Sanofi Pasteur (ActHib), and contained HibPRP-T 10μg. ActHib was consisted of a pre-filled diluent syringe (0.5 mL) and a 1-dose vial of lyophilized powder. The vaccine batch number was E9567–1, and it was valid until 30 Apr, 2012.

Safety evaluation

Safety observations were performed by the investigators in all subjects at 30 min and at 6, 24, 48 and 72 h after vaccinations. Symptoms such as redness, induration, skin rash, fever, vomiting, abnormal crying, lethargy, loss of appetite and irritability were assessed and recorded. In addition, adverse events that occurred in the period from days 4 to 28 after each vaccine dose were also recorded (parents/guardians were instructed to call the study center if adverse events were experienced).

Immunogenicity evaluation

200 μL blood samples were obtained from subjects before vaccination and at 28 d after completion of the immunization schedule. Serum Hib-PRP antibody concentrations pre- and post-immunization were determined by enzyme-linked immunosorbent assay (ELISA). A concentration ≥ 1.0 μg/mL was adopted as the threshold for long-term protection, while ≥ 0.15 μg/mL was adopted as the threshold for natural protection.

Statistical analysis

The proportions of infants in the study groups with Hib-PRP antibody concentrations ≥ 1.0 μg/mL and ≥ 0.15 μg/mL after immunization were determined. Vaccine safety was evaluated via the incidence rate of all post-vaccination local and systemic adverse reactions.

Epidata software was used for data input and SPSS 16.0 statistical software was applied for data processing. Comparisons between long-term protection and natural protection rates were tested by χ² and t-tests by comparing antibody geometric mean concentrations (GMCs) in the study groups. The incidence rate of adverse reactions among the study groups was inspected by a χ² test, with an inspection level of α = 0.05.

Abbreviations:
ELISA	=	enzyme-linked immunosorbent assay
GMC	=	geometric mean concentration
Hib	=	Haemophilus influenzae type b
PRP	=	polyribosylribitol phosphate

Disclosure of potential conflicts of interest

The authors declare no potential conflicts of interest.

Sources of support

This study was supported by Sanofi Pasteur S.A.

10.4161/hv.25526