- Email: [email protected]
Protective potency of recombinant meningococcal IgA1 protease and its structural derivatives upon animal invasion with meningococcal and pneumococcal infections
Accepted Manuscript Protective potency of recombinant meningococcal IgA1 protease and its structural derivatives upon animal invasion with meningococcal and pneumococcal infections Olga Kotelnikova, Alexander Alliluev, Alexei Zinchenko, Larisa Zhigis, Yuri Prokopenko, Elena Nokel, Olga Razgulyaeva, Vera Zueva, Marina Tokarskaya, Natalia Yastrebova, Elena Gordeeva, Tatyana Melikhova, Elena Kaliberda, Lev Rumsh PII:
S1286-4579(19)30025-5
DOI:
https://doi.org/10.1016/j.micinf.2019.02.003
Reference:
MICINF 4626
To appear in:
Microbes and Infection
Received Date: 20 June 2018 Revised Date:
6 February 2019
Accepted Date: 11 February 2019
Please cite this article as: O. Kotelnikova, A. Alliluev, A. Zinchenko, L. Zhigis, Y. Prokopenko, E. Nokel, O. Razgulyaeva, V. Zueva, M. Tokarskaya, N. Yastrebova, E. Gordeeva, T. Melikhova, E. Kaliberda, L. Rumsh, Protective potency of recombinant meningococcal IgA1 protease and its structural derivatives upon animal invasion with meningococcal and pneumococcal infections, Microbes and Infection, https:// doi.org/10.1016/j.micinf.2019.02.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
ACCEPTED MANUSCRIPT
Protective potency of recombinant meningococcal IgA1 protease and its
1 2
structural derivatives upon animal invasion with meningococcal and
3
pneumococcal infections
5
RI PT
4
Olga Kotelnikovaa., Alexander Alliluevb, Alexei Zinchenkoa, Larisa Zhigisa*, Yuri Prokopenkoa, Elena Nokela, Olga Razgulyaevaa, Vera Zuevaa, Marina Tokarskayac, Natalia
7
Yastrebovac, Elena Gordeevaa, Tatyana Melikhovaa, Elena Kaliberdaa, Lev Rumsha
SC
6
8
10 11
a
Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of
M AN U
9
Sciences, ul. Miklukho-Maklaya 16/10, Moscow, 117997 Russia b
Central Research Institute of Epidemiology of the Federal Service on Customers’
Rights Protection and Human Well-Being Surveillance, ul. Novogireevskaya 3a, Moscow,
13
111123 Russia
14
c
15
Mechnikov Research Institute for Vaccines and Sera, Malyi Kazennyi per. 5a,
Moscow, 105064 Russia
EP
16
*Corresponding author. Fax: +7(495)335-08-12; e-mail address: [email protected];
AC C
17
TE D
12
18
postal address: Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian
19
Academy of Sciences, ul. Miklukho-Maklaya 16/10, Moscow, 117997, Russia.
20 21 22
Abstract Immunization of mice with recombinant IgA1 protease of Neisseria meningitidis or
23
several structural derivatives thereof protects the animals infected with a variety of deadly
24
pathogens, including N. meningitidis serogroups A, B, and C and 3 serotypes of Streptococcus
2
ACCEPTED MANUSCRIPT pneumonia. In sera of rabbits immunized with inactivated pneumococcal cultures, antibodies
26
binding IgA1-protease from N. meningitidis serogroup B were detected. Thus, the cross-
27
reactive protection against meningococcal and pneumococcal infections has been
28
demonstrated in vivo. Presumably it indicates the presence of common epitopes in the N.
29
meningitidis IgA1 protease and S. pneumoniae surface proteins.
30
RI PT
25
Keywords: IgA1 protease; Neisseria meningitidis; Streptococcus pneumoniae; Meningococcal
32
vaccines; Epitopes
SC
31
34
M AN U
33
1. Introduction
35
According to the WHO, meningitis caused by Streptococcus pneumoniae (S.
37
pneumoniae) and Neisseria meningitidis (N. meningitidis) accounts for more than 70% of
38
meningitis cases and is regarded as severe, often fatal illness. For prevention of both
39
pneumococcal and meningococcal infections, vaccines based on capsular polysaccharides are
40
currently used, with the exception of the vaccine against meningococcus serogroup B. The
41
latter is a multicomponent vaccine based on surface proteins of this bacterium [1–4].
EP
TE D
36
Implementation of pneumococcal and meningococcal vaccines reduced the incidence
43
of these infections [4, 5]. At the same time, the number of disease cases caused by serotypes
44
of S. pneumoniae not included in the existing vaccines increased. In addition, the use of these
45
pneumococcal vaccines is limited by the low immunogenicity of capsular polysaccharides,
46
high phenotypic variability, and genetic plasticity of pneumococci [6]. In this regard,
47
development of new pneumococcal vaccines based on capsular polysaccharides seems to be
48
of little promise. Moreover, most currently used vaccines are narrow-spectrum ones, targeting
49
a particular pathogen. To provide protection from a wide variety of circulating and
AC C
42
3
ACCEPTED MANUSCRIPT 50
continuously mutating strains of both meningococcus and pneumococcus, a multivalent
51
vaccine employing a common virulence factor is required.
52
At present, bacterial IgA1 proteases (IgA1pr) are considered as potential vaccine candidates. Serine type endopeptidases of Gram-negative bacteria (N. meningitidis, H.
54
influenzae, N. gonorrhoae), as well as Zn2 + metalloproteinases of Gram-positive (S.
55
pneumoniae, S.sanguis, S. oralis) bacteria, are important virulence factors of these microbes
56
[7–10]. Despite the different catalytic mechanisms and low homology of the primary
57
structures of IgA1 proteases of Gram-negative and Gram-positive bacteria, these enzymes
58
specifically bind and cleave serum and secretory human immunoglobulins A1 at the hinge
59
region of heavy chain [8, 11, 12]. Cleavage of secretory immunoglobulin A1 in mucous
60
membranes of the host organism promotes bacterial adhesion to epithelial cells, colonization
61
of tissues, and further development of the infection process [8, 13].
SC
M AN U
Previously, we have shown that serine type IgA1pr from a living culture of N.
TE D
62
RI PT
53
meningitidis serogroup A [14] and several variants of recombinant IgA1 protease of N.
64
meningitidis serogroup B: M1K2–N963LEH6, MA28–N963LEH6 and MA28-P1004LEH6, as well as
65
some structural derivatives of IgA1pr, for example, ME135–H328LEH6, induced formation of
66
specific antibodies, supporting up to 80% of mice survival upon infection with live virulent
67
culture of meningococci of the major epidemic serogroups A, B, and C [15–18]. Other
68
authors demonstrated the potential of using streptococcal metallo-IgA1pr as a surface-
69
protective antigen to protect animals from diseases caused by pneumococci and Streptococcus
70
suis serotype 2 [9, 19]. Despite the limited number of common epitopes in serine-type IgA1pr
71
and metallo-IgA1pr [20, 21], we assumed that IgA1pr of meningococci may have a protective
72
effect against pneumococcal infection.
AC C
EP
63
73
The aim of this work was to evaluate the protective properties of recombinant
74
meningococcal IgA1pr and structural derivatives and to investigate in vivo the ability of these
4
ACCEPTED MANUSCRIPT 75
proteins to protect animals from pneumococcal and meningococcal infections and the
76
possibility of cross-immune protection.
77
2. Materials and methods
RI PT
78 79 80
2.1. Antigens
SC
81
Primary structure of recombinant IgA1pr MA28–P1004LEH6 with MM of about 109
83
kDa is a fragment of a full-sized serine IgA protease of N. meningitidis serogroup B strain
84
H44/76 and contains only the protease domain and gamma-peptide of the full-length enzyme
85
[8, 10, 21]. The numbering of amino acid residues in structural formulas of all the
86
recombinant proteins used in this work corresponds to that of sequence of full-length IgA
87
protease from N. meningitidis serogroup B H44/76 in
88
http://www.ncbi.nlm.nih.gov/protein/ADC80147. IgA1pr MA28–P1004LEH6 was chosen as the
89
base structure for creating shorter recombinant proteins due to the high homology of its amino
90
acid sequence to that of neisserial IgA proteases [20, 21].
TE D
EP
91
M AN U
82
Recombinant IgA1pr from N. meningitidis serogroup В strain Н44/76 with the primary structure MA28–P1004LEH6 (MM ~109 kDa) and its N-terminal fragment (Protein I, ME135–
93
H328LEH6, MM 23.4 kDa), were obtained as described in detail earlier [17, 18]. Proteins II
94
and III are fusion recombinant proteins containing three or two fragments from different
95
regions of base molecule IgA1pr MA28–P1004LEH6 (Protein II, MM 59.4 kDa, Protein III,
96
MM 34.7 kDa) obtained by a similar technique. Protein II contains fragments from the N-
97
terminal, central, and C-terminal region of the base molecule MA28-P1004LEH6, Protein III
98
contains fragments from the N-terminal and C-terminal regions of the base molecule. All
AC C
92
5
ACCEPTED MANUSCRIPT 99 100
investigated recombinant proteins contained the LEH6 sequence in the C-terminal part of molecules. According to SDS-PAGE, purity of the proteins was >93%.
101
2.2. Bacterial cultures
RI PT
102 103 104
N. meningitidis serogroup A (strain A208), serogroup В (strain Н44/76), and
serogroup C (strain C0638) were obtained from the Scientific Centre for Expert Evaluation of
106
Medicinal Products (Ministery of Healthcare of the Russian Federation); S. pneumoniae
107
serotypes 3, 6А, 6В, 7F, 9N, 14, 15B, 18С, 19А, and 19F, from Microbiology Laboratory of
108
the National Medical Research Center of Children’s Health (Ministery of Healthcare of the
109
Russian Federation).
M AN U
SC
105
110
2.3. Immunogenic and protective activities of recombinant IgA1pr MA28–P1004LEH6 and
112
Proteins I, II, and III
113
TE D
111
Immunogenic and protective activities of the preparations were assessed using female
115
BALB/c mice (16–18 g, 3–4 months old, 7 animals per group) and chinchilla rabbits (3–4 kg,
116
4 animals per group). The animals were immunized with IgA1pr of N. meningitidis or its
117
structural derivatives by intravenous injection twice with an interval of 45 days at a dose of 40
118
µg per mouse and 1400 µg per rabbit. Levels of specific antibodies against IgA1pr of N.
119
meningitidis and its structural derivatives were estimated on day 30 after the first
120
immunization and day 12 after the second immunization. A standard solid-phase ELISA with
121
peroxidase-conjugated goat anti-mouse or anti-rabbit antibodies (Biosciences Pharmingen)
122
and sorption of the optimal doses of each antigen on the plate was used [15, 16].
AC C
EP
114
6
ACCEPTED MANUSCRIPT 123
One month after the second immunization, mice were infected with live virulent
124
cultures of N. meningitidis serogroups A, B, or C as described in [15, 16] or S. pneumoniae
125
serotypes 3, 14, or 19A (see below). To infect mice, suspension of lyophilized S. pneumoniae culture in physiological saline
RI PT
126
was inoculated onto Petri dishes with solid medium (blood agar with 5% defibrinated bovine
128
blood) and incubated at 37 °C under 5% СО2 for 20 h. Then, bacterial suspension was
129
transferred onto fresh Petri dish and incubated at 37 °C under 5% СО2 for 18 h. Mice were
130
infected intraperitoneally with 100 microbial cells in 0.5 ml per mouse. Four hours after
131
infection, blood samples were drawn from retro-orbital sinus and diluted 1 : 4 with
132
physiological saline; 10-µl aliquots were spread on Petri dishes containing growth medium.
133
The number of colonies was registered 18 h after blood inoculation.
M AN U
SC
127
134
2.4. Passive protection of mice against meningococcal infection with anti-pneumococcal sera
TE D
135 136
Hyperimmune sera for passive protection were obtained as described earlier [22] from
138
rabbits immunized with inactivated S. pneumoniae serotypes 3, 6A, 14, 18C, and 19A. Naive
139
(not-immunized) recipient mice were intravenously injected with 0.2 ml serum per mouse.
140
Total serum of rabbits immunized with IgA1pr MA28-P1004LEH6 was used as a positive
141
control and total serum from the same rabbits before immunization, as a negative control.
142
Three hours later, the animals were infected with live virulent culture of N. meningitidis
143
serogroup B strain Н44/76. The CFU number was accessed as described above, the number of
144
deceased animals was registered on day 5 after infection [16].
AC C
EP
137
145 146 147
2.5. Statistical analysis
7
ACCEPTED MANUSCRIPT 148
Statistical processing of the results was carried out using Probit analysis and Student’s
149
t-test in the MS Office Excel software. Results are presented as mean values ± standard
150
deviation for p ≤ 0.05.
152
RI PT
151
3. Results and discussion
153
After a single immunization of both mice and rabbits with the base IgA1pr MA28-
SC
154
P1004LEH6 or Proteins I, II, and III, antibody titers for all used preparations did not differ
156
significantly from this parameter in control animals injected with isotonic NaCl solution.
157
After the second immunization of animals, intensive production of antibodies interacting with
158
IgA1pr MA28-P1004LEH6, was registered (see Table 1) indicating formation of immunological
159
memory. Immunogenicity of Proteins II and III did not differ significantly from
160
immunogenicity of IgA1pr MA28-P1004LEH6, while immunogenicity of Protein I was
161
significantly lower.
164 165
Table 1
AC C
163
EP
162
TE D
M AN U
155
Protective properties of all preparations were evaluated in a model of infection of
166
immunized mice with live virulent cultures of N. meningitidis serogroups A, B, and C, as well
167
as S. pneumoniae serotypes 3, 14, and 19A, which were chosen for testing as the most
168
common virulent pneumococcal serotypes. Infecting mice with various pathogens, we aimed
169
to demonstrate in principle the possibility to protect mammals by preparations based on
170
meningococcal serogroup B IgA1pr from infection of a different etiology. The protection
171
against different strains was different, which was most likely determined by their virulence.
8
ACCEPTED MANUSCRIPT 172
Therefore, the comparative assessment of preparations was conducted independently, in the
173
first instance, for each pathogen (Fig. 1).
174
Fig. 1
RI PT
175 176 177
All preparations provided protection against N. meningitidis in 50 to 80% mice (Fig. 1a).In addition, despite the limited homology of the primary structures between IgA1
179
proteases of N. meningitidis and S. pneumoniae, immunization of mice with preparations
180
based on meningococcal IgA1pr showed pronounced protective effect upon direct infection
181
with live pneumococcal culture (Fig. 1b). In case of S. pneumoniae serotypes 3, 14, and 19A
182
infection, level of bacteremia was significantly lowered in mice immunized with
183
meningococcal IgA1pr: CFU numbers in these animals were 48, 56, and 59% to control
184
value, respectively. This demonstrated the principal ability of IgA1pr of N. meningitidis to
185
protect mice from fatal infection with not only meningococcal, but also pneumococcal live
186
cultures. Presumably, the protective effect was exhibited because of the presence in
187
meningococcal IgA1pr of conformational antigenic determinants similar to that in
188
pneumococcal surface proteins.
M AN U
TE D
EP
As for Proteins I, II, and III, they also protected mice against pneumococcal infection,
AC C
189
SC
178
190
but the degree of protection varied. When mice were infected with pneumococcal serotype
191
19A, protection by all Proteins was minimal (CFU numbers were 65–89% to control).
192
Maximum protective activity was demonstrated by Proteins II and III. Protective effect of
193
these preparations against serotype 14 was comparable to that produced against meningococci
194
of all three serogroups: CFU numbers were approximately 30%. This effect even exceeded
195
the effect of immunization with base IgA1pr (56%). The analogous result was observed for
196
Protein III in relation to pneumococcal serotype 3. Immunization with Protein I did not result
9
ACCEPTED MANUSCRIPT in significant protection against pneumococci, while it provided high protection level (CFU of
198
40% to control and lower) in mice infected with all three meningococcal serogroups. This
199
indicates the absence of epitopes common to pneumococcal proteins in the structure of
200
Protein I.
201
RI PT
197
To determine the presence of antigenic determinants common to both meningococcal IgA1pr and pneumococci of different serotypes, we assessed the anti-pneumococcal rabbit
203
sera interactions with meningococcal IgA1pr MA28-P1004LEH6 or Proteins I, II and III (Table
204
1). The total serum of rabbits immunized with N. meningitidis IgA1pr MA28-P1004LEH6 was
205
used as a positive control.
M AN U
206
SC
202
In sera of rabbits immunized with inactivated S. pneumoniae of various serotypes, antibodies binding IgA1pr MA28-P1004LEH6 and Proteins I, II and III were detected. It was
208
shown that the level of these antibodies was significantly lower than after immunization with
209
meningococcal IgA1pr MA28-P1004LEH6 and Proteins I, II, and III. Depending on structure of
210
tested proteins and the pneumococcus serotype, the antibody titer varied from 1 : 25 to 1:
211
3200. It should be noted that the lowest level of antibodies binding to IgA1pr MA28-
212
P1004LEH6 and Proteins I, II, and III in all rabbit sera studied was observed for Protein I,
213
which correlated with the lack of protection against pneumococcal infection for this protein,
214
as described above (Fig. 1b). This is probably due to specific features of the ME135-H328LEH6
215
structure [17, 18].
EP
AC C
216
TE D
207
As shown in the Table 1, after immunization of rabbits with pneumococcus serotype
217
19A, the level of antibodies interacting with meningococcal IgA1pr MA28-P1004LEH6 was
218
sufficiently lower than for serotypes 3 and 14. Thus, weak protection of IgA1pr-immunized
219
animals against S. pneumoniae serotype 19A (Fig. 1b) can be explained by lower content of
220
antibodies recognizing antigenic determinants common to meningococcus IgA1pr and
10
ACCEPTED MANUSCRIPT 221
pneumococcal surface proteins in anti-serotype 19A serum than in the case of serotypes 3 and
222
14. To demonstrate the cross-reactivity in vivo, the protective activity of rabbit sera was
224
assessed by passive protection of recipient mice, i.e. by bacteremia levels in these mice 4 h
225
after infection with live virulent culture of N. meningitidis serogroup B and the number of
226
deceased animals on day 5 after infection (Fig. 2). The rabbits were immunized with
227
inactivated cultures of the most common virulent strains 3, 6А, 14, 18С, and 19А, included in
228
the PPSV23 anti-pneumococcal vaccine, or with meningococcal IgA1pr MA28-P1004LEH6.
229
The latter serum was used as a reference preparation in assessing the protective role of
230
specific antibodies to N. meningitidis IgA1pr against meningococcal infection (Fig. 2b).
231
Injection of this serum with homologous anti-IgA1pr antibody titer of 1 : 10,240 in serial two-
232
fold dilutions resulted in a clear dependence of the protective effect on the antibody level
233
(Fig. 2a), which indicates active participation of these antibodies in protection.
TE D
M AN U
SC
RI PT
223
Four hours after infection of mice having received whole anti-pneumococcal sera with
235
the culture of N. meningitidis serogroup B, blood CFU levels were between 49 and 76% to the
236
value in mice having received a non-immune serum (100%). Therefore, correlation between
237
blood CFU and the level of antibodies binding IgA1pr of N. meningitidis was poor. Stronger
238
correlation was observed between anti-IgA1pr antibody titer and the number of deceased mice
239
(20 to 60%), suggesting active involvement of IgA1pr-binding antibodies in protection
240
against meningococcal infection. At the same time it cannot be excluded that the
241
protective effect may be caused not only by the above mentioned antibodies, but also by
242
antibodies binding other meningococcal surface components.
AC C
EP
234
243 244 245
Fig. 2.
11
ACCEPTED MANUSCRIPT 246
Our study reveals that immunization of animals with IgA1pr MA28-P1004LEH6 or Proteins I, II, III provides the formation of immunological memory and can protect against
248
both meningococcal and a number of pneumococcal fatal infections. Protective properties of
249
meningococcal IgA1pr MA28-P1004LEH6 and Proteins I, II, III against pneumococci may be
250
attributed to their conformational epitopes close in structure to epitopes of pneumococcal
251
surface proteins. In the model of passive animal protection, sera from rabbits immunized with
252
S. pneumoniae of different serotypes were shown to be capable of animal protection from
253
infection with N. meningitidis, at least serogroup B. Thus, we conclude that pneumococcal
254
and meningococcal infections can cause a cross-protective effect. This makes meningococcal
255
IgA1 proteases and their recombinant derivatives the potential components of polyvalent
256
vaccines. In the future, one of the tested preparations can be used as the base of a
257
monocomponent multivalent vaccine to protect against not only N. meningitidis, but also
258
against a wide range of other pathogens (N. gonorrhoeae, H. influenzae, S. pneumoniae)
259
carrying similar antigenic determinants. Results presented in this article show the possibility
260
in principle of creating such vaccines.
TE D
M AN U
SC
RI PT
247
263 264 265
Conflict of interests
The authors declare no conflicts of interest.
AC C
262
EP
261
Acknowledgements
We thank Dr. L. D. Papusheva, the Head of Experimental biological laboratory,
266
Central Research Institute of Epidemiology of the Federal Service on Customers' Rights
267
Protection and Human Well-Being Surveillance for assistance in determining the
268
immunogenicity of the test preparations in animal experiments.
269 270
References
12
ACCEPTED MANUSCRIPT 1. Tomczyk S, Bennett NM, Stoecker C, Gierke R, Moore MR, Whitney CG, et al. Use of 13-
272
valent pneumococcal conjugate vaccine and 23-valent pneumococcal polysaccharide vaccine
273
among adults aged ≥65 years: recommendations of the Advisory Committee on Immunization
274
Practices (ACIP). MMWR Morb Mortal Wkly Rep 2014;63:822-5.
275
2. Bogaert D, Sluijter M, De Groot R, Hermans PW. Multiplex opsonophagocytosis assay
276
(MOPA): a useful tool for the monitoring of the 7-valent pneumococcal conjugate vaccine.
277
Vaccine 2004;22:4014-20.
278
3. Pace D, Pollard AJ. Meningococcal A, C, Y and W-135 polysaccharide-protein conjugate
279
vaccines. Arch Dis Child 2007;92:909-15.
280
4. Crum-Cianflone N, Sullivan E. Meningococcal vaccinations. Infect Dis Ther 2016;5:89–
281
112.
282
5. Leventer-Roberts M, Feldman BS, Brufman I, Cohen-Stavi CJ, Hoshen M, Balicer RD.
283
Effectiveness of 23-valent pneumococcal polysaccharide vaccine against invasive disease and
284
hospital-treated pneumonia among people aged ≥65 years: a retrospective case-control study.
285
Clin Infect Dis 2015;60:1472-80.
286
6. De Paolis F, Beghetto E, Spadoni A, Montagnani F, Felici F, Oggioni MR, et al.
287
Identification of a human immunodominant B-cell epitope within the immunoglobulin A1
288
protease of Streptococcus pneumoniae. BMC Microbiology 2007;7:113.
289
7. Henderson IR, Nataro JP. Virulence functions of autotransporter proteins. Infect Immun
290
2001;69:1231–43.
291
8. Mistry D, Stockley RA. IgA1 protease. Intern Int J Biochem Cell Biol
292
2006;38:1244–8.
293
9. Lei F, Zhao J, Lin L, Zhang Q, Xu Z, Han L, et al. Characterization of IgA1 protease
294
as surface protective antigen of Streptococcus suis serotype 2. Microbes Infect
295
2016;18:285–9.
AC C
EP
TE D
M AN U
SC
RI PT
271
13
ACCEPTED MANUSCRIPT 10. Roussel-Jazede V, Arenas J, Langereis JD, Tommassen J, van Ulsen P. Variable
297
processing of the IgA protease autotransporter at the cell surface of Neisseria
298
meningitidis. Microbiology 2014;160:2421–31.
299
11. Plaut AG. The IgA1 proteases of pathogenic bacteria. Annu Rev Microbiol 1983;37:603–
300
22.
301
12. Weiser JN, Bae D, Fasching C, Scamurra RW, Ratner AJ, Janoff EN. Antibody-enhanced
302
pneumococcal adherence requires IgA1 protease. Proc Natl Acad Sci USA 2003;100:4215–
303
20.
304
13. Kilian M, Reincholdt J, Lomcholt H, Poulsen K, Frandsen EV. Biological significance of
305
IgA1 proteases in bacterial colonization and pathogenesis: critical evaluation of experimental
306
evidence. APMIS (Copenhagen) 1996;104:321-38.
307
14. Yagudaeva EYu, Zhigis LS, Razguliaeva OA, Zueva VS, Mel'nikov EE, Zubov VP, et al.
308
Isolation and determination of the activity of IgA1 protease from Neisseria meningitidis. Rus
309
J Bioorg Chem 2010;36:81–9.
310
15. Kotel'nikova OV, Alliluev AP, Drozhzhina EYu, Koroleva IS, Sitnikova EA, Zinchenko
311
AA, et al. Protective properties of recombinant IgA1 protease from meningococcus.
312
Biochemistry (Moscow) Suppl Ser B 2013;7:305-10.
313
16. Kotelnikova OV, Zinchenko AA, Vikhrov AA, Alliluev AP, Serova OV, Gordeeva EA, et
314
al. Serological assessment of immunogenic properties of recombinant proteins based on
315
meningococcus protease IgA1. Bull Exp Biol Med 2016;161:391-4.
316
17. Zinchenko AA, Alliluev AP, Serova OP, Gordeeva EA, Zhigis LS, Zueva VS, et al.
317
Immunogenic and protective properties of recombinant proteins based on meningococcal
318
IgA1 protease. J Meningitis 2015;1,1000102.
AC C
EP
TE D
M AN U
SC
RI PT
296
14
ACCEPTED MANUSCRIPT 18. Zinchenko AA, Kotelnikova OV, Gordeeva EA, Prokopenko YuA, Razgulyaeva OA,
320
Serova OV, et al. Immunogenic and protective properties of Neisseria meningitidis IgA1
321
protease and of its truncated fragments. Rus J Bioorg Chem 2018;44:64-72.
322
19. Janoff EN, Rubins JB, Fasching C, Charboneau D, Rahkola JT, Plaut AG, et al.
323
Pneumococcal IgA1 protease subverts specific protection by human IgA1. Mucosal Immunol
324
2014;7: 249–56.
325
20. Lomholt H, Kilian M. Antigenic relationships among immunoglobulin A1 proteases from
326
Haemophilus, Neisseria, and Streptococcus species. Infect Immun. 1994;62:3178-83.
327
21. Lomholt H, Poulsen K and Killian M. Comparative characterization of the iga gene
328
encoding IgA protease in Neisseria meningitidis, Neisseria gonorrhoeae and Haemophilus
329
influenzae. Mol Microbiol.1995;15:495-506.
330
22. Elkina SI, Tokarskaya MM, Yastrebova NE, Aparin PG. Method of obtaining
331
pneumococcal hyperimmune sera. Patent RU 2624871 C1,
332
https://worldwide.espacenet.com/publicationDetails/biblio?II=0&ND=3&adjacent=true&loca
333
le=en_EP&FT=D&date=20170707&CC=RU&NR=2624871C1&KC=C1
334
EP
TE D
M AN U
SC
RI PT
319
Figure legends
336
Fig. 1. The level of bacteremia in mice immunized with N. meningitidis IgA1pr MA28-
337
P1004LEH6 and Proteins I, II, III after infection with live cultures of meningococcus
338
serogroups A, B, and C (a) or pneumococcus serotypes 3, 14, and 19A (b). The figures shown
339
above the bars report average numbers of CFU (%) of three replicates; the error bars indicate
340
standard deviations for p≤0.05.
341
AC C
335
15
ACCEPTED MANUSCRIPT Fig. 2. Protective properties of sera from rabbits immunized with IgA1pr MA28-P1004LEH6 (a)
343
and with inactivated S. pneumoniae cultures of various serotypes (b), after recipient mice
344
infection with live virulent culture of N. meningitidis serogroup B. The data shown are means
345
of three replicates; the error bars indicate standard deviations for p≤0.05.
RI PT
342
AC C
EP
TE D
M AN U
SC
346
ACCEPTED MANUSCRIPT
Table 1. The level (1/titer) of antibodies binding to N. meningitidis IgA1pr MA28– P1004LEH6 and Proteins I, II, III in sera of rabbits immunized with N. meningitidis IgA1pr MA28– P1004LEH6 or with inactivated cultures of S. pneumoniae of various serotypes
15B 200 40 3.5 25 5 4.9 70 10 4.8 70 10 4.8
M AN U
SC
RI PT
Levels of antibodies in sera of immunized rabbits Antigen N. meningitidis S. pneumoniae serotypes IgA1pr* 6А 14 18С 19А 9N 6В 7F 3 IgA1pr* 11,520 1600 200 1120 1040 360 1280 400 180 3 221 320 40 160 240 101 452 80 50 ± 3.1 3.5 3.2 3.2 3.4 3.15 3.4 3.5 t** 280 240 180 30 70 90 Protein I 1600 180 50 320 50 10 40 46 50 6 10 25 ± 4.4 4.8 4.1 4.2 3.5 4.9 4.8 4.7 t Protein II 4480 1440 100 1120 280 360 1600 200 180 640 402 20 160 40 101 320 40 50 ± 4.0 6.8 5.1 4.1 3.4 3.06 4.3 3.5 t Protein III 8960 3200 90 1120 400 280 560 180 100 1280 640 25 160 80 40 80 50 20 ± 4.0 4.7 5.1 3.4 4.1 6.5 3.5 6.9 t 28 1004 *IgA1pr MA –P LEH6
AC C
EP
TE D
**The t criterion was calculated with relation to antibody titer in animals immunized with IgA1 protease, Protein I, Protein II, or Protein III, respectively. At t> 2.3, the difference is considered reliable.
19F 180 50 3.5 35 5 4.9 240 46 4.2 90 25 4.7
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
S1286-4579(19)30025-5
DOI:
https://doi.org/10.1016/j.micinf.2019.02.003
Reference:
MICINF 4626
To appear in:
Microbes and Infection
Received Date: 20 June 2018 Revised Date:
6 February 2019
Accepted Date: 11 February 2019
Please cite this article as: O. Kotelnikova, A. Alliluev, A. Zinchenko, L. Zhigis, Y. Prokopenko, E. Nokel, O. Razgulyaeva, V. Zueva, M. Tokarskaya, N. Yastrebova, E. Gordeeva, T. Melikhova, E. Kaliberda, L. Rumsh, Protective potency of recombinant meningococcal IgA1 protease and its structural derivatives upon animal invasion with meningococcal and pneumococcal infections, Microbes and Infection, https:// doi.org/10.1016/j.micinf.2019.02.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
ACCEPTED MANUSCRIPT
Protective potency of recombinant meningococcal IgA1 protease and its
1 2
structural derivatives upon animal invasion with meningococcal and
3
pneumococcal infections
5
RI PT
4
Olga Kotelnikovaa., Alexander Alliluevb, Alexei Zinchenkoa, Larisa Zhigisa*, Yuri Prokopenkoa, Elena Nokela, Olga Razgulyaevaa, Vera Zuevaa, Marina Tokarskayac, Natalia
7
Yastrebovac, Elena Gordeevaa, Tatyana Melikhovaa, Elena Kaliberdaa, Lev Rumsha
SC
6
8
10 11
a
Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of
M AN U
9
Sciences, ul. Miklukho-Maklaya 16/10, Moscow, 117997 Russia b
Central Research Institute of Epidemiology of the Federal Service on Customers’
Rights Protection and Human Well-Being Surveillance, ul. Novogireevskaya 3a, Moscow,
13
111123 Russia
14
c
15
Mechnikov Research Institute for Vaccines and Sera, Malyi Kazennyi per. 5a,
Moscow, 105064 Russia
EP
16
*Corresponding author. Fax: +7(495)335-08-12; e-mail address: [email protected];
AC C
17
TE D
12
18
postal address: Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian
19
Academy of Sciences, ul. Miklukho-Maklaya 16/10, Moscow, 117997, Russia.
20 21 22
Abstract Immunization of mice with recombinant IgA1 protease of Neisseria meningitidis or
23
several structural derivatives thereof protects the animals infected with a variety of deadly
24
pathogens, including N. meningitidis serogroups A, B, and C and 3 serotypes of Streptococcus
2
ACCEPTED MANUSCRIPT pneumonia. In sera of rabbits immunized with inactivated pneumococcal cultures, antibodies
26
binding IgA1-protease from N. meningitidis serogroup B were detected. Thus, the cross-
27
reactive protection against meningococcal and pneumococcal infections has been
28
demonstrated in vivo. Presumably it indicates the presence of common epitopes in the N.
29
meningitidis IgA1 protease and S. pneumoniae surface proteins.
30
RI PT
25
Keywords: IgA1 protease; Neisseria meningitidis; Streptococcus pneumoniae; Meningococcal
32
vaccines; Epitopes
SC
31
34
M AN U
33
1. Introduction
35
According to the WHO, meningitis caused by Streptococcus pneumoniae (S.
37
pneumoniae) and Neisseria meningitidis (N. meningitidis) accounts for more than 70% of
38
meningitis cases and is regarded as severe, often fatal illness. For prevention of both
39
pneumococcal and meningococcal infections, vaccines based on capsular polysaccharides are
40
currently used, with the exception of the vaccine against meningococcus serogroup B. The
41
latter is a multicomponent vaccine based on surface proteins of this bacterium [1–4].
EP
TE D
36
Implementation of pneumococcal and meningococcal vaccines reduced the incidence
43
of these infections [4, 5]. At the same time, the number of disease cases caused by serotypes
44
of S. pneumoniae not included in the existing vaccines increased. In addition, the use of these
45
pneumococcal vaccines is limited by the low immunogenicity of capsular polysaccharides,
46
high phenotypic variability, and genetic plasticity of pneumococci [6]. In this regard,
47
development of new pneumococcal vaccines based on capsular polysaccharides seems to be
48
of little promise. Moreover, most currently used vaccines are narrow-spectrum ones, targeting
49
a particular pathogen. To provide protection from a wide variety of circulating and
AC C
42
3
ACCEPTED MANUSCRIPT 50
continuously mutating strains of both meningococcus and pneumococcus, a multivalent
51
vaccine employing a common virulence factor is required.
52
At present, bacterial IgA1 proteases (IgA1pr) are considered as potential vaccine candidates. Serine type endopeptidases of Gram-negative bacteria (N. meningitidis, H.
54
influenzae, N. gonorrhoae), as well as Zn2 + metalloproteinases of Gram-positive (S.
55
pneumoniae, S.sanguis, S. oralis) bacteria, are important virulence factors of these microbes
56
[7–10]. Despite the different catalytic mechanisms and low homology of the primary
57
structures of IgA1 proteases of Gram-negative and Gram-positive bacteria, these enzymes
58
specifically bind and cleave serum and secretory human immunoglobulins A1 at the hinge
59
region of heavy chain [8, 11, 12]. Cleavage of secretory immunoglobulin A1 in mucous
60
membranes of the host organism promotes bacterial adhesion to epithelial cells, colonization
61
of tissues, and further development of the infection process [8, 13].
SC
M AN U
Previously, we have shown that serine type IgA1pr from a living culture of N.
TE D
62
RI PT
53
meningitidis serogroup A [14] and several variants of recombinant IgA1 protease of N.
64
meningitidis serogroup B: M1K2–N963LEH6, MA28–N963LEH6 and MA28-P1004LEH6, as well as
65
some structural derivatives of IgA1pr, for example, ME135–H328LEH6, induced formation of
66
specific antibodies, supporting up to 80% of mice survival upon infection with live virulent
67
culture of meningococci of the major epidemic serogroups A, B, and C [15–18]. Other
68
authors demonstrated the potential of using streptococcal metallo-IgA1pr as a surface-
69
protective antigen to protect animals from diseases caused by pneumococci and Streptococcus
70
suis serotype 2 [9, 19]. Despite the limited number of common epitopes in serine-type IgA1pr
71
and metallo-IgA1pr [20, 21], we assumed that IgA1pr of meningococci may have a protective
72
effect against pneumococcal infection.
AC C
EP
63
73
The aim of this work was to evaluate the protective properties of recombinant
74
meningococcal IgA1pr and structural derivatives and to investigate in vivo the ability of these
4
ACCEPTED MANUSCRIPT 75
proteins to protect animals from pneumococcal and meningococcal infections and the
76
possibility of cross-immune protection.
77
2. Materials and methods
RI PT
78 79 80
2.1. Antigens
SC
81
Primary structure of recombinant IgA1pr MA28–P1004LEH6 with MM of about 109
83
kDa is a fragment of a full-sized serine IgA protease of N. meningitidis serogroup B strain
84
H44/76 and contains only the protease domain and gamma-peptide of the full-length enzyme
85
[8, 10, 21]. The numbering of amino acid residues in structural formulas of all the
86
recombinant proteins used in this work corresponds to that of sequence of full-length IgA
87
protease from N. meningitidis serogroup B H44/76 in
88
http://www.ncbi.nlm.nih.gov/protein/ADC80147. IgA1pr MA28–P1004LEH6 was chosen as the
89
base structure for creating shorter recombinant proteins due to the high homology of its amino
90
acid sequence to that of neisserial IgA proteases [20, 21].
TE D
EP
91
M AN U
82
Recombinant IgA1pr from N. meningitidis serogroup В strain Н44/76 with the primary structure MA28–P1004LEH6 (MM ~109 kDa) and its N-terminal fragment (Protein I, ME135–
93
H328LEH6, MM 23.4 kDa), were obtained as described in detail earlier [17, 18]. Proteins II
94
and III are fusion recombinant proteins containing three or two fragments from different
95
regions of base molecule IgA1pr MA28–P1004LEH6 (Protein II, MM 59.4 kDa, Protein III,
96
MM 34.7 kDa) obtained by a similar technique. Protein II contains fragments from the N-
97
terminal, central, and C-terminal region of the base molecule MA28-P1004LEH6, Protein III
98
contains fragments from the N-terminal and C-terminal regions of the base molecule. All
AC C
92
5
ACCEPTED MANUSCRIPT 99 100
investigated recombinant proteins contained the LEH6 sequence in the C-terminal part of molecules. According to SDS-PAGE, purity of the proteins was >93%.
101
2.2. Bacterial cultures
RI PT
102 103 104
N. meningitidis serogroup A (strain A208), serogroup В (strain Н44/76), and
serogroup C (strain C0638) were obtained from the Scientific Centre for Expert Evaluation of
106
Medicinal Products (Ministery of Healthcare of the Russian Federation); S. pneumoniae
107
serotypes 3, 6А, 6В, 7F, 9N, 14, 15B, 18С, 19А, and 19F, from Microbiology Laboratory of
108
the National Medical Research Center of Children’s Health (Ministery of Healthcare of the
109
Russian Federation).
M AN U
SC
105
110
2.3. Immunogenic and protective activities of recombinant IgA1pr MA28–P1004LEH6 and
112
Proteins I, II, and III
113
TE D
111
Immunogenic and protective activities of the preparations were assessed using female
115
BALB/c mice (16–18 g, 3–4 months old, 7 animals per group) and chinchilla rabbits (3–4 kg,
116
4 animals per group). The animals were immunized with IgA1pr of N. meningitidis or its
117
structural derivatives by intravenous injection twice with an interval of 45 days at a dose of 40
118
µg per mouse and 1400 µg per rabbit. Levels of specific antibodies against IgA1pr of N.
119
meningitidis and its structural derivatives were estimated on day 30 after the first
120
immunization and day 12 after the second immunization. A standard solid-phase ELISA with
121
peroxidase-conjugated goat anti-mouse or anti-rabbit antibodies (Biosciences Pharmingen)
122
and sorption of the optimal doses of each antigen on the plate was used [15, 16].
AC C
EP
114
6
ACCEPTED MANUSCRIPT 123
One month after the second immunization, mice were infected with live virulent
124
cultures of N. meningitidis serogroups A, B, or C as described in [15, 16] or S. pneumoniae
125
serotypes 3, 14, or 19A (see below). To infect mice, suspension of lyophilized S. pneumoniae culture in physiological saline
RI PT
126
was inoculated onto Petri dishes with solid medium (blood agar with 5% defibrinated bovine
128
blood) and incubated at 37 °C under 5% СО2 for 20 h. Then, bacterial suspension was
129
transferred onto fresh Petri dish and incubated at 37 °C under 5% СО2 for 18 h. Mice were
130
infected intraperitoneally with 100 microbial cells in 0.5 ml per mouse. Four hours after
131
infection, blood samples were drawn from retro-orbital sinus and diluted 1 : 4 with
132
physiological saline; 10-µl aliquots were spread on Petri dishes containing growth medium.
133
The number of colonies was registered 18 h after blood inoculation.
M AN U
SC
127
134
2.4. Passive protection of mice against meningococcal infection with anti-pneumococcal sera
TE D
135 136
Hyperimmune sera for passive protection were obtained as described earlier [22] from
138
rabbits immunized with inactivated S. pneumoniae serotypes 3, 6A, 14, 18C, and 19A. Naive
139
(not-immunized) recipient mice were intravenously injected with 0.2 ml serum per mouse.
140
Total serum of rabbits immunized with IgA1pr MA28-P1004LEH6 was used as a positive
141
control and total serum from the same rabbits before immunization, as a negative control.
142
Three hours later, the animals were infected with live virulent culture of N. meningitidis
143
serogroup B strain Н44/76. The CFU number was accessed as described above, the number of
144
deceased animals was registered on day 5 after infection [16].
AC C
EP
137
145 146 147
2.5. Statistical analysis
7
ACCEPTED MANUSCRIPT 148
Statistical processing of the results was carried out using Probit analysis and Student’s
149
t-test in the MS Office Excel software. Results are presented as mean values ± standard
150
deviation for p ≤ 0.05.
152
RI PT
151
3. Results and discussion
153
After a single immunization of both mice and rabbits with the base IgA1pr MA28-
SC
154
P1004LEH6 or Proteins I, II, and III, antibody titers for all used preparations did not differ
156
significantly from this parameter in control animals injected with isotonic NaCl solution.
157
After the second immunization of animals, intensive production of antibodies interacting with
158
IgA1pr MA28-P1004LEH6, was registered (see Table 1) indicating formation of immunological
159
memory. Immunogenicity of Proteins II and III did not differ significantly from
160
immunogenicity of IgA1pr MA28-P1004LEH6, while immunogenicity of Protein I was
161
significantly lower.
164 165
Table 1
AC C
163
EP
162
TE D
M AN U
155
Protective properties of all preparations were evaluated in a model of infection of
166
immunized mice with live virulent cultures of N. meningitidis serogroups A, B, and C, as well
167
as S. pneumoniae serotypes 3, 14, and 19A, which were chosen for testing as the most
168
common virulent pneumococcal serotypes. Infecting mice with various pathogens, we aimed
169
to demonstrate in principle the possibility to protect mammals by preparations based on
170
meningococcal serogroup B IgA1pr from infection of a different etiology. The protection
171
against different strains was different, which was most likely determined by their virulence.
8
ACCEPTED MANUSCRIPT 172
Therefore, the comparative assessment of preparations was conducted independently, in the
173
first instance, for each pathogen (Fig. 1).
174
Fig. 1
RI PT
175 176 177
All preparations provided protection against N. meningitidis in 50 to 80% mice (Fig. 1a).In addition, despite the limited homology of the primary structures between IgA1
179
proteases of N. meningitidis and S. pneumoniae, immunization of mice with preparations
180
based on meningococcal IgA1pr showed pronounced protective effect upon direct infection
181
with live pneumococcal culture (Fig. 1b). In case of S. pneumoniae serotypes 3, 14, and 19A
182
infection, level of bacteremia was significantly lowered in mice immunized with
183
meningococcal IgA1pr: CFU numbers in these animals were 48, 56, and 59% to control
184
value, respectively. This demonstrated the principal ability of IgA1pr of N. meningitidis to
185
protect mice from fatal infection with not only meningococcal, but also pneumococcal live
186
cultures. Presumably, the protective effect was exhibited because of the presence in
187
meningococcal IgA1pr of conformational antigenic determinants similar to that in
188
pneumococcal surface proteins.
M AN U
TE D
EP
As for Proteins I, II, and III, they also protected mice against pneumococcal infection,
AC C
189
SC
178
190
but the degree of protection varied. When mice were infected with pneumococcal serotype
191
19A, protection by all Proteins was minimal (CFU numbers were 65–89% to control).
192
Maximum protective activity was demonstrated by Proteins II and III. Protective effect of
193
these preparations against serotype 14 was comparable to that produced against meningococci
194
of all three serogroups: CFU numbers were approximately 30%. This effect even exceeded
195
the effect of immunization with base IgA1pr (56%). The analogous result was observed for
196
Protein III in relation to pneumococcal serotype 3. Immunization with Protein I did not result
9
ACCEPTED MANUSCRIPT in significant protection against pneumococci, while it provided high protection level (CFU of
198
40% to control and lower) in mice infected with all three meningococcal serogroups. This
199
indicates the absence of epitopes common to pneumococcal proteins in the structure of
200
Protein I.
201
RI PT
197
To determine the presence of antigenic determinants common to both meningococcal IgA1pr and pneumococci of different serotypes, we assessed the anti-pneumococcal rabbit
203
sera interactions with meningococcal IgA1pr MA28-P1004LEH6 or Proteins I, II and III (Table
204
1). The total serum of rabbits immunized with N. meningitidis IgA1pr MA28-P1004LEH6 was
205
used as a positive control.
M AN U
206
SC
202
In sera of rabbits immunized with inactivated S. pneumoniae of various serotypes, antibodies binding IgA1pr MA28-P1004LEH6 and Proteins I, II and III were detected. It was
208
shown that the level of these antibodies was significantly lower than after immunization with
209
meningococcal IgA1pr MA28-P1004LEH6 and Proteins I, II, and III. Depending on structure of
210
tested proteins and the pneumococcus serotype, the antibody titer varied from 1 : 25 to 1:
211
3200. It should be noted that the lowest level of antibodies binding to IgA1pr MA28-
212
P1004LEH6 and Proteins I, II, and III in all rabbit sera studied was observed for Protein I,
213
which correlated with the lack of protection against pneumococcal infection for this protein,
214
as described above (Fig. 1b). This is probably due to specific features of the ME135-H328LEH6
215
structure [17, 18].
EP
AC C
216
TE D
207
As shown in the Table 1, after immunization of rabbits with pneumococcus serotype
217
19A, the level of antibodies interacting with meningococcal IgA1pr MA28-P1004LEH6 was
218
sufficiently lower than for serotypes 3 and 14. Thus, weak protection of IgA1pr-immunized
219
animals against S. pneumoniae serotype 19A (Fig. 1b) can be explained by lower content of
220
antibodies recognizing antigenic determinants common to meningococcus IgA1pr and
10
ACCEPTED MANUSCRIPT 221
pneumococcal surface proteins in anti-serotype 19A serum than in the case of serotypes 3 and
222
14. To demonstrate the cross-reactivity in vivo, the protective activity of rabbit sera was
224
assessed by passive protection of recipient mice, i.e. by bacteremia levels in these mice 4 h
225
after infection with live virulent culture of N. meningitidis serogroup B and the number of
226
deceased animals on day 5 after infection (Fig. 2). The rabbits were immunized with
227
inactivated cultures of the most common virulent strains 3, 6А, 14, 18С, and 19А, included in
228
the PPSV23 anti-pneumococcal vaccine, or with meningococcal IgA1pr MA28-P1004LEH6.
229
The latter serum was used as a reference preparation in assessing the protective role of
230
specific antibodies to N. meningitidis IgA1pr against meningococcal infection (Fig. 2b).
231
Injection of this serum with homologous anti-IgA1pr antibody titer of 1 : 10,240 in serial two-
232
fold dilutions resulted in a clear dependence of the protective effect on the antibody level
233
(Fig. 2a), which indicates active participation of these antibodies in protection.
TE D
M AN U
SC
RI PT
223
Four hours after infection of mice having received whole anti-pneumococcal sera with
235
the culture of N. meningitidis serogroup B, blood CFU levels were between 49 and 76% to the
236
value in mice having received a non-immune serum (100%). Therefore, correlation between
237
blood CFU and the level of antibodies binding IgA1pr of N. meningitidis was poor. Stronger
238
correlation was observed between anti-IgA1pr antibody titer and the number of deceased mice
239
(20 to 60%), suggesting active involvement of IgA1pr-binding antibodies in protection
240
against meningococcal infection. At the same time it cannot be excluded that the
241
protective effect may be caused not only by the above mentioned antibodies, but also by
242
antibodies binding other meningococcal surface components.
AC C
EP
234
243 244 245
Fig. 2.
11
ACCEPTED MANUSCRIPT 246
Our study reveals that immunization of animals with IgA1pr MA28-P1004LEH6 or Proteins I, II, III provides the formation of immunological memory and can protect against
248
both meningococcal and a number of pneumococcal fatal infections. Protective properties of
249
meningococcal IgA1pr MA28-P1004LEH6 and Proteins I, II, III against pneumococci may be
250
attributed to their conformational epitopes close in structure to epitopes of pneumococcal
251
surface proteins. In the model of passive animal protection, sera from rabbits immunized with
252
S. pneumoniae of different serotypes were shown to be capable of animal protection from
253
infection with N. meningitidis, at least serogroup B. Thus, we conclude that pneumococcal
254
and meningococcal infections can cause a cross-protective effect. This makes meningococcal
255
IgA1 proteases and their recombinant derivatives the potential components of polyvalent
256
vaccines. In the future, one of the tested preparations can be used as the base of a
257
monocomponent multivalent vaccine to protect against not only N. meningitidis, but also
258
against a wide range of other pathogens (N. gonorrhoeae, H. influenzae, S. pneumoniae)
259
carrying similar antigenic determinants. Results presented in this article show the possibility
260
in principle of creating such vaccines.
TE D
M AN U
SC
RI PT
247
263 264 265
Conflict of interests
The authors declare no conflicts of interest.
AC C
262
EP
261
Acknowledgements
We thank Dr. L. D. Papusheva, the Head of Experimental biological laboratory,
266
Central Research Institute of Epidemiology of the Federal Service on Customers' Rights
267
Protection and Human Well-Being Surveillance for assistance in determining the
268
immunogenicity of the test preparations in animal experiments.
269 270
References
12
ACCEPTED MANUSCRIPT 1. Tomczyk S, Bennett NM, Stoecker C, Gierke R, Moore MR, Whitney CG, et al. Use of 13-
272
valent pneumococcal conjugate vaccine and 23-valent pneumococcal polysaccharide vaccine
273
among adults aged ≥65 years: recommendations of the Advisory Committee on Immunization
274
Practices (ACIP). MMWR Morb Mortal Wkly Rep 2014;63:822-5.
275
2. Bogaert D, Sluijter M, De Groot R, Hermans PW. Multiplex opsonophagocytosis assay
276
(MOPA): a useful tool for the monitoring of the 7-valent pneumococcal conjugate vaccine.
277
Vaccine 2004;22:4014-20.
278
3. Pace D, Pollard AJ. Meningococcal A, C, Y and W-135 polysaccharide-protein conjugate
279
vaccines. Arch Dis Child 2007;92:909-15.
280
4. Crum-Cianflone N, Sullivan E. Meningococcal vaccinations. Infect Dis Ther 2016;5:89–
281
112.
282
5. Leventer-Roberts M, Feldman BS, Brufman I, Cohen-Stavi CJ, Hoshen M, Balicer RD.
283
Effectiveness of 23-valent pneumococcal polysaccharide vaccine against invasive disease and
284
hospital-treated pneumonia among people aged ≥65 years: a retrospective case-control study.
285
Clin Infect Dis 2015;60:1472-80.
286
6. De Paolis F, Beghetto E, Spadoni A, Montagnani F, Felici F, Oggioni MR, et al.
287
Identification of a human immunodominant B-cell epitope within the immunoglobulin A1
288
protease of Streptococcus pneumoniae. BMC Microbiology 2007;7:113.
289
7. Henderson IR, Nataro JP. Virulence functions of autotransporter proteins. Infect Immun
290
2001;69:1231–43.
291
8. Mistry D, Stockley RA. IgA1 protease. Intern Int J Biochem Cell Biol
292
2006;38:1244–8.
293
9. Lei F, Zhao J, Lin L, Zhang Q, Xu Z, Han L, et al. Characterization of IgA1 protease
294
as surface protective antigen of Streptococcus suis serotype 2. Microbes Infect
295
2016;18:285–9.
AC C
EP
TE D
M AN U
SC
RI PT
271
13
ACCEPTED MANUSCRIPT 10. Roussel-Jazede V, Arenas J, Langereis JD, Tommassen J, van Ulsen P. Variable
297
processing of the IgA protease autotransporter at the cell surface of Neisseria
298
meningitidis. Microbiology 2014;160:2421–31.
299
11. Plaut AG. The IgA1 proteases of pathogenic bacteria. Annu Rev Microbiol 1983;37:603–
300
22.
301
12. Weiser JN, Bae D, Fasching C, Scamurra RW, Ratner AJ, Janoff EN. Antibody-enhanced
302
pneumococcal adherence requires IgA1 protease. Proc Natl Acad Sci USA 2003;100:4215–
303
20.
304
13. Kilian M, Reincholdt J, Lomcholt H, Poulsen K, Frandsen EV. Biological significance of
305
IgA1 proteases in bacterial colonization and pathogenesis: critical evaluation of experimental
306
evidence. APMIS (Copenhagen) 1996;104:321-38.
307
14. Yagudaeva EYu, Zhigis LS, Razguliaeva OA, Zueva VS, Mel'nikov EE, Zubov VP, et al.
308
Isolation and determination of the activity of IgA1 protease from Neisseria meningitidis. Rus
309
J Bioorg Chem 2010;36:81–9.
310
15. Kotel'nikova OV, Alliluev AP, Drozhzhina EYu, Koroleva IS, Sitnikova EA, Zinchenko
311
AA, et al. Protective properties of recombinant IgA1 protease from meningococcus.
312
Biochemistry (Moscow) Suppl Ser B 2013;7:305-10.
313
16. Kotelnikova OV, Zinchenko AA, Vikhrov AA, Alliluev AP, Serova OV, Gordeeva EA, et
314
al. Serological assessment of immunogenic properties of recombinant proteins based on
315
meningococcus protease IgA1. Bull Exp Biol Med 2016;161:391-4.
316
17. Zinchenko AA, Alliluev AP, Serova OP, Gordeeva EA, Zhigis LS, Zueva VS, et al.
317
Immunogenic and protective properties of recombinant proteins based on meningococcal
318
IgA1 protease. J Meningitis 2015;1,1000102.
AC C
EP
TE D
M AN U
SC
RI PT
296
14
ACCEPTED MANUSCRIPT 18. Zinchenko AA, Kotelnikova OV, Gordeeva EA, Prokopenko YuA, Razgulyaeva OA,
320
Serova OV, et al. Immunogenic and protective properties of Neisseria meningitidis IgA1
321
protease and of its truncated fragments. Rus J Bioorg Chem 2018;44:64-72.
322
19. Janoff EN, Rubins JB, Fasching C, Charboneau D, Rahkola JT, Plaut AG, et al.
323
Pneumococcal IgA1 protease subverts specific protection by human IgA1. Mucosal Immunol
324
2014;7: 249–56.
325
20. Lomholt H, Kilian M. Antigenic relationships among immunoglobulin A1 proteases from
326
Haemophilus, Neisseria, and Streptococcus species. Infect Immun. 1994;62:3178-83.
327
21. Lomholt H, Poulsen K and Killian M. Comparative characterization of the iga gene
328
encoding IgA protease in Neisseria meningitidis, Neisseria gonorrhoeae and Haemophilus
329
influenzae. Mol Microbiol.1995;15:495-506.
330
22. Elkina SI, Tokarskaya MM, Yastrebova NE, Aparin PG. Method of obtaining
331
pneumococcal hyperimmune sera. Patent RU 2624871 C1,
332
https://worldwide.espacenet.com/publicationDetails/biblio?II=0&ND=3&adjacent=true&loca
333
le=en_EP&FT=D&date=20170707&CC=RU&NR=2624871C1&KC=C1
334
EP
TE D
M AN U
SC
RI PT
319
Figure legends
336
Fig. 1. The level of bacteremia in mice immunized with N. meningitidis IgA1pr MA28-
337
P1004LEH6 and Proteins I, II, III after infection with live cultures of meningococcus
338
serogroups A, B, and C (a) or pneumococcus serotypes 3, 14, and 19A (b). The figures shown
339
above the bars report average numbers of CFU (%) of three replicates; the error bars indicate
340
standard deviations for p≤0.05.
341
AC C
335
15
ACCEPTED MANUSCRIPT Fig. 2. Protective properties of sera from rabbits immunized with IgA1pr MA28-P1004LEH6 (a)
343
and with inactivated S. pneumoniae cultures of various serotypes (b), after recipient mice
344
infection with live virulent culture of N. meningitidis serogroup B. The data shown are means
345
of three replicates; the error bars indicate standard deviations for p≤0.05.
RI PT
342
AC C
EP
TE D
M AN U
SC
346
ACCEPTED MANUSCRIPT
Table 1. The level (1/titer) of antibodies binding to N. meningitidis IgA1pr MA28– P1004LEH6 and Proteins I, II, III in sera of rabbits immunized with N. meningitidis IgA1pr MA28– P1004LEH6 or with inactivated cultures of S. pneumoniae of various serotypes
15B 200 40 3.5 25 5 4.9 70 10 4.8 70 10 4.8
M AN U
SC
RI PT
Levels of antibodies in sera of immunized rabbits Antigen N. meningitidis S. pneumoniae serotypes IgA1pr* 6А 14 18С 19А 9N 6В 7F 3 IgA1pr* 11,520 1600 200 1120 1040 360 1280 400 180 3 221 320 40 160 240 101 452 80 50 ± 3.1 3.5 3.2 3.2 3.4 3.15 3.4 3.5 t** 280 240 180 30 70 90 Protein I 1600 180 50 320 50 10 40 46 50 6 10 25 ± 4.4 4.8 4.1 4.2 3.5 4.9 4.8 4.7 t Protein II 4480 1440 100 1120 280 360 1600 200 180 640 402 20 160 40 101 320 40 50 ± 4.0 6.8 5.1 4.1 3.4 3.06 4.3 3.5 t Protein III 8960 3200 90 1120 400 280 560 180 100 1280 640 25 160 80 40 80 50 20 ± 4.0 4.7 5.1 3.4 4.1 6.5 3.5 6.9 t 28 1004 *IgA1pr MA –P LEH6
AC C
EP
TE D
**The t criterion was calculated with relation to antibody titer in animals immunized with IgA1 protease, Protein I, Protein II, or Protein III, respectively. At t> 2.3, the difference is considered reliable.
19F 180 50 3.5 35 5 4.9 240 46 4.2 90 25 4.7
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT