Anti-chlamydial IgG Neutralizing Ability in Nonzoonotic Atypical Community Acquired Respiratory Tract Infections

Abstract

Chlamydophila pneumoniae is a pathogenic agent, involved in various types of infection. This study has evaluated the ability of IgG antibodies in outpatient, with acute respiratory tract infections from C. pneumoniae, to neutralize in vitro purified elementary bodies of this bacterium, revealing a good neutralizing performance of IgG antibodies.

Chlamydophila pneumoniae is an atypical bacterium: first isolation dates back to 1965 [1], established as a human respiratory pathogen in 1986 [2], and potential zoonotic transmission described in 2010 [1]. C. pneumoniae is an obligate intracellular bacterium, which is detected from 5 to 10 % of community-acquired pneumonia (CAP) [3]. Moreover, C. pneumoniae is involved in various types of infection (sub-acute, acute, and chronic). Acute disease from this bacterium is most common [4]. Seroprevalence increases with age, e.g. in young it is about 50 %, while in elderly about 75 % [4]. Despite some pathological conditions have been attributed to C. pneumoniae [5–7], this bacterium frequently causes mild or asymptomatic infections, which often remain unrecognized and consequently untreated [4]. For these reasons this study has evaluated the ability of IgG antibodies from outpatient, with acute C. pneumoniae infection, to neutralize in vitro purified elementary bodies (EBs) of this pathogenic agent. We have studied 50 non consecutive outpatients in 2012–2013 with acute infection by C. pneumoniae, with following clinical, imaging and laboratory data: nonproductive cough, low-grade fever, and radiological images in suspicion of interstitial pneumonia, and positivity of real time-polymerase chain reaction (RT-PCR) and/or serological techniques, as described previously [8]. From these patients we have collected 200 sera samples. The sera were selected as follows: 50 at the same time of acute infection diagnosis (1st set), 50 after 2 weeks (2nd set), 50 after 12 weeks (3th set) and 50 after 20 weeks (4th set). In addition, to evaluate specificity of this assay, 25 sera are studied as control: 10 sera positive for antibodies anti-Mycoplasma pneumoniae, 5 with antibodies anti-Legionella pneumophila, and 10 negative sera for antibodies against C. pneumoniae, M. pneumoniae and L. pneumophila. The sera positive against C. pneumoniae are tested also against purified EBs of Chlamydophila psittaci and Chlamydia trachomatis to evaluate the specificity of neutralising antibodies against C. pneumoniae. The software and criteria used to perform statistical analysis were described previously [9]. The microimmunofluorescence (MIF) assay was used to detect and measure the titre of IgG antibodies anti-C. pneumoniae (strain IOL-207) in the sera (Fig. 1). This assay and the diagnostic criteria of acute/past infection used in this work are described earlier [8]. A sucrose gradient was used to obtain purified EBs (titre 5 × 10⁵ inclusion forming units/mL) of C. pneumoniae (strain IOL-207) and to perform neutralizing test [10]. Rabbit serum was used as a source of complement (5 % final concentration). The sera anti-C. pneumoniae were inactivated at 56 °C for 30 min and diluted 1:5 in Hanks balanced salt solution (HBSS) and then 100 μL were distributed in triplicate over 96-well microtitre plates. EBs were diluted in 0.25 M sucrose–10 mM potassium phosphate–5 mM glutamic acid (SPG), to obtain 2 × 10⁴ inclusion forming units/mL, and 90 μL was added to prediluted sera and to 100 μL of HBSS to control. The mixtures (serum and EBs) and control (HBSS and EBs), incubated for 30 min at 37 °C, were then inoculated in triplicate onto monolayers of a continuous LLC-MK2 cell line derived from Rhesus monkey kidney tissue grown in 24-well plates containing a glass coverslip at the bottom [10]. Subsequently, 800 μL of chlamydial growth medium (Eagle’s minimum essential medium with 10 % heat-inactivated fetal calf serum, 5 µg/mL of glucose, 1 µg/mL of cycloheximide) was added to obtain a final serum dilution of 1:10. After centrifugation at 1000 g for 1 h, the monolayers were incubated at 37 °C for 48 h, fixed in methanol and then stained with a fluorescein-conjugated monoclonal antibody specific for the chlamydial LPS (Meridian, Cincinnati, USA). An average was taken and the results were expressed as reduction percentage of inclusion forming units/mL from control monolayers. A ≤50 % reduction of inclusion forming units/mL from control was defined as neutralizing. Moreover, in order to obtain a neutralizing titre, two dilutions (1:20 and 1:40) were performed. In addition, neutralisation reactions using purified EBs of genovar D of Chlamydia trachomatis and 6BC of Chlamydophila psittaci were performed to control the specificity of neutralising antibodies anti-C. pneumoniae The targets of immune response have been highlighted using an immunoblot analysis. Briefly, proteins were separated by SDS-PAGE using 12 % (w/v) precast gel (Invitrogen, Paisley, UK) and transferred to nitrocellulose membranes (Sigma, St. Louis, USA). These membranes were blotted with the 50 positive sera of 4th set, diluted 1/100. Chlamydia-specific bands were detected with peroxidase-labelled goat anti-human IgG (Dako, Glostrup, Denmark) diluted 1:500, as the secondary antibody and with 4-chloro-naphthol (Bio-Rad, Milan, Italy) for colorimetric visualization. Table 1 shows the distribution of IgG titre in sera from patients with acute infection by C. pneumoniae. The 40 % of the 1st and 2nd sera set did not show IgG antibodies, which appear more late (12 weeks). High titres of IgG are reached in 4th set (20 weeks), although 40 % of the sera of the 3th set has a titre of IgG from 256 to 512. Table 1 exhibit the neutralizing ability of IgG, with average neutralizing activity (percentage) and modal titre value. The neutralizing activity increases in direct proportion to increase antibody titer, but also increasing IgG age (Table 1). Only 24 % of 1st sera set shows neutralizing activity, with low average percentage (58 %) and low modal titre (1:10) (Table 1). On the contrary, the 3th and 4th set of sera exhibit excellent neutralizing activity with very high values (average percentage 89.4 % and titre modal value 1:40) (Table 1). All sera from patients with acute C. pneumoniae infection, in the absence of IgG show no neutralizing activity (IgG titre <32 in 1st and 2nd sera sets) (Table 1). The immunoblot has showed a reactivity of sera against the 40, 100 and 120 kDa proteins (Fig. 2).

Fig. 1.

Bright apple-green staining due to reaction between immuno-complexes (IgG and C. pneumoniae EBs) and a fluorescein-conjugated monoclonal antibody anti-human IgG

Table 1.

IgG activity anti-C. pneumoniae

IgG perform from 1st set of sera				IgG perform from 2nd set of sera				IgG perform from 3th set of sera				IgG perform from 4th set of sera
No. (%) of sera	IgG titre	Neutral. activitya (%)	Neutral. titreb	No. (%) of sera	IgG titre	Neutral. activitya (%)	Neutral. titreb	No. (%) of sera	IgG titre	Neutral. activitya (%)	Neutral. titreb	No. (%) of sera	IgG titre	Neutral. activitya (%)	Neutral. titreb
38 (76)	1:32	<50	–c	32 (64)	1:32	<50	–c	5 (10)	1:32	65	1:10	0 (0)	–c	–d	–d
12 (24)	1:64	58	1:10	3 (6)	1:64	62	1:10	10 (20)	1:64	65	1:10	0 (0)	–c	–d	–d
0 (0)	–c	–d	–d	15 (30)	1:128	68	1:10e	15 (30)	1:128	90	1:20	1 (2)	1:128	100	1:40
0 (0)	–c	–d	–d	0 (0)	–c	–d	–d	18 (36)	1:256	95	1:40e	28 (56)	1:256	100	1:40
0 (0)	–c	–d	–d	0 (0)	–c	–d	–d	2 (4)	1:512	95	1:40e	16 (32)	1:512	100	1:40
0 (0)	–c	–d	–d	0 (0)	–c	–d	–d	0	–c	–d	–d	5 (10)	1:1024	100	1:40

^aNeutral. activity: neutralizing activity; ^b Neutral. titre: neutralizing titre; ^c not revealed; ^d not performed; ^e referring to the mode (statistically the most frequent value)

Fig. 2.

Reactivity of IgG against Chlamydophila pneumoniae EBs revealed by immuno-blot technique. M marker; 1 positive serum sample (titre 1:64)

In addition, IgM anti-C. pneumoniae were detected in 90 % of 1st set and in 100 % of 2nd set of samples. The 25 sera, used as controls, are all negative for detection of antibodies anti-C. pneumoniae and show no neutralizing activity against C. pneumoniae EBs (p < 0.001). Furthermore, the sera show no neutralizing activity against EBs of C. trachomatis and C. psittaci (p < 0.001). Therefore the assay specificity has been excellent (Fig. 1). These data display that this assay is not limited by cross-reactivity among Chlamydiae, strict human pathogens. This work has shown in vitro an excellent activity of IgG antibodies, titre and time-dependent, to neutralize C. pneumoniae EBs. These results may explain why this bacterium frequently causes infections often unrecognized and untreated, despite some pathological conditions attributed to that bacterium [4]. In vivo further studies are needed to clarify the neutralizing ability of IgG and to understand their real role played in acute and specially in chronic infections (e.g. atherosclerosis, primary biliary cirrhosis, neurological disorders, etc.) and to develop a vaccine [5–7, 11, 12]. In addition, for the high specificity the neutralization assay, although laborious to perform [13], could be suggested, as second diagnostic level, in indirect laboratory diagnosis of C. pneumoniae infections to avoid cross-reactivity of some serological techniques [13, 14]; moreover, the neutralizing test could be applied as a diagnostic method to assess the onset time of the chlamydial infection. In conclusion, simultaneous use of direct and indirect diagnostic techniques, as described for other pathogenic agent [8, 15], permit to obtain the best diagnostic results, assaying different diagnostic methodologies and understanding the best applicable technologies to the diagnosis of the infectious diseases.

Abbreviations

CAP

Community-acquired pneumonia

EBs

Elementary bodies

HBSS

Hanks balanced salt solution

MIF

Microimmunofluorescence

RT-PCR

Real time-polymerase chain reaction

SPG

Sucrose–potassium phosphate–glutamic acid